Methods to treat conditions associated with insulin signaling dysregulation

ABSTRACT

The invention discloses a method to identify proteins involved in the insulin signaling pathway. The invention also discloses suitable targets for the development of new therapeutics to treat, prevent or ameliorate pathological conditions associated with dysregulation of the insulin signaling pathway. The invention also relates to methods to treat, prevent or ameliorate said conditions and pharmaceutical compositions therefor as well as to a method to identify compounds with therapeutic usefulness to treat pathological conditions associated with dysregulation of the insulin signaling pathway.

FIELD OF THE INVENTION

This invention relates to a method to identify proteins involved in theinsulin signaling pathway, methods for identifying compounds useful totreat pathological conditions associated with dysregulation of theinsulin signaling pathway as well as to methods and pharmaceuticalcompositions to treat, prevent or ameliorate conditions associated withdysregulation of the insulin signaling pathway.

BACKGROUND OF THE INVENTION

The regulation of numerous cellular processes involves the transmissionof extracelluar information through the cell via a network of associatedproteins. Defects in signal transduction mechanisms may be associatedwith specific diseases, for example, Type II or noninsulin-dependentdiabetes mellitus (NIDDM) is thought to be due largely due to defects inthe insulin signalling pathway.

The molecular mechanisms of insulin signalling have been extensivelyreviewed. (Avruch, J. (1998). Mol Cell Biochem 182, 31-48;Combettes-Souverain, M., and Issad, T. (1998). Diabetes Metab 24,477-89; Kahn, B. B. (1998) Cell 92, 593-6; Virkamaki, A., et al. (1999).J Clin Invest 103, 931-43). Recent genetic studies in C. elegans andDrosophila have shown a remarkable conservation of the insulin pathwaysin invertebrates and mammals (Paradis, S. et al. (1999) Genes Dev 13,1438-52; Weinkove, D., and Leevers, S. J. (2000). Curr Opin Genet Dev10, 75-80). In addition, many aspects of the insulin pathway areconserved between humans and Drosophila. For example, almost allcurrently known genes involved in the insulin signaling pathway inhumans are also found in Drosophila and have similar biochemicalfunctions.

Using Drosophila as a model system, Applicants herein disclose a methodto identify proteins involved in the insulin signalling pathway.Employing said method, Applicants have discovered and describe hereinseveral new proteins involved in the insulin signalling pathway. It iscontemplated herein that these proteins and the genes encoding saidproteins may serve as drug targets for the development of therapeuticsto treat, prevent or ameliorate diabetes and other pathologicalconditions associated with dysregulation of the insulin signallingpathway.

SUMMARY OF THE INVENTION

The instant application discloses a method to employ transgenicDrosophila to identify proteins involved in the insulin signallingpathway. Human homologs of the Drosophila genes identified according tothis method are suitable targets for the development of new therapeuticsto treat, prevent or ameliorate pathological conditions associated withthe dysregulation of the insulin signaling pathway. Thus, in one aspectthe invention relates to a method to identify modulators useful to treator ameliorate said conditions, including Type II diabetes and the Type Asyndrome of insulin resistance comprising a) assaying for the ability ofa candidate modulator to modulate the biochemical function of a proteinselected from the group consisting of those disclosed in Table 4 orTable 5 and/or modulate gene expression of said protein and which canfurther include b) assaying for the ability of an identified modulatorto reverse the pathological effects observed in animal models ofpathological conditions associated with the dysregulation of the insulinsignaling pathway and/or in clinical studies with subjects with saidconditions.

In another aspect, the invention relates to a method to treat, preventor ameliorate pathological conditions associated with the dysregulationof the insulin signaling pathway, including Type II diabetes and theType A syndrome of insulin resistance, comprising administering to asubject in need thereof an effective amount of a modulator of a proteinselected from the group consisting of those disclosed in Table 4 orTable 5, wherein said modulator, e.g., inhibits or enhances thebiochemical function of said protein. In a further embodiment, themodulator comprises antibodies to said protein or fragments thereof,wherein said antibodies can inhibit the biochemical function of saidprotein in said subject.

In another embodiment the modulator inhibits or enhances the geneexpression of a protein selected from the group consisting of thosedisclosed in Table 4 or Table 5. In a further embodiment, the modulatorcomprises any one or more substances selected from the group consistingof antisense oligonucleotides, triple helix DNA, ribozymes, RNAaptamers, siRNA and double or single stranded RNA wherein saidsubstances are designed to inhibit gene expression of said protein.

In another aspect, the invention relates to a method to treat, preventor ameliorate pathological conditions associated with dysregulation ofthe insulin signaling pathway, including Type II diabetes and the Type Asyndrome of insulin resistance, comprising administering to a subject inneed thereof a pharmaceutical composition comprising an effective amountof a modulator of a protein selected from the group consisting of thosedisclosed in Table 4 or Table 5. In various embodiments, saidpharmaceutical composition comprises antibodies to said protein orfragments thereof, wherein said antibodies can inhibit the biochemicalfunction of said protein in said subject and/or any one or moresubstances selected from the group consisting of antisenseoligonucleotides, triple helix DNA, ribozymes, RNA aptamers, siRNA anddouble or single stranded RNA wherein said substances are designed toinhibit gene expression of said protein.

In another aspect, the invention relates to a pharmaceutical compositioncomprising a modulator to a protein selected from the group consistingof those disclosed in Table 4 or Table 5 in an amount effective totreat, prevent or ameliorate a pathological condition associated withdysregulation of the insulin signaling pathway, including Type IIdiabetes and the Type A syndrome of insulin resistance, in a subject inneed thereof. In one embodiment, said modulator may e.g., inhibit orenhance the biochemical functions of said protein. In a furtherembodiment said modulator comprises antibodies to said protein orfragments thereof, wherein said antibodies can, e.g., inhibit thebiochemical functions of said protein.

In a further embodiment, said pharmaceutical composition comprises amodulator which may e.g., inhibit or enhance gene expression of saidprotein. In a further embodiment, said modulator comprises any one ormore substances selected from the group consisting of antisenseoligonucleotides, triple helix DNA, ribozymes, RNA aptamers, siRNA ordouble or single stranded RNA directed to a nucleic acid sequence ofsaid protein wherein said substances are designed to inhibit geneexpression of said protein.

In another aspect, the invention relates to a method to diagnosesubjects suffering from pathological conditions associated withdysregulation of the insulin signaling pathway who may be suitablecandidates for treatment with modulators to a protein selected from thegroup consisting of those disclosed in Table 4 or Table 5 comprisingdetecting levels of any one or more of said proteins in a biologicalsample from said subject wherein subjects with altered levels comparedto controls would be suitable candidates for modulator treatment.

In another aspect, the invention relates to a method to diagnosesubjects suffering from pathological conditions associated withdysregulation of the insulin signaling pathway who may be suitablecandidates for treatment with modulators to a protein selected from thegroup consisting of those disclosed in Table 4 or Table 5 comprisingassaying mRNA levels of any one or more of said protein in a biologicalsample from said subject wherein subjects with altered levels comparedto controls would be suitable candidates for modulator treatment.

In yet another aspect, there is provided a method to treat, prevent orameliorate pathological conditions associated with dysregulation of theinsulin signaling pathway, including Type II diabetes and the Type Asyndrome of insulin resistance comprising: (a) assaying for mRNA and/orprotein levels of a protein selected from the group consisting of thosedisclosed in Table 4 or Table 5 in a subject; and (b) administering to asubject with altered levels of mRNA and/or protein levels compared tocontrols a modulator to said protein in an amount sufficient to treat,prevent or ameliorate the pathological effects of said condition. Inparticular embodiments, said modulator inhibits or enhances thebiochemical function of said protein or gene expression of said protein.

In yet another aspect of the present invention there are provided assaymethods and diagnostic kits comprising the components necessary todetect mRNA levels or protein levels of any one or more proteinsselected from the group consisting of those disclosed in Table 4 orTable 5 in a biological sample, said kit comprising e.g. polynucleotidesencoding any one or more proteins selected from the group consisting ofthose disclosed in Table 4 or Table 5; nucleotide sequencescomplementary to said protein; any one or more of said proteins, orfragments thereof of antibodies that bind to any one or more of saidproteins, or to fragments thereof. In a preferred embodiment, such kitsalso comprise instructions detailing the procedures by which the kitcomponents are to be used.

The present invention also pertains to the use of a modulator to aprotein selected from the group consisting of those disclosed in Table 4or Table 5 in the manufacture of a medicament for the treatment,prevention or amelioration of pathological conditions associated withdysregulation of the insulin signaling pathway, including Type IIdiabetes and the Type A syndrome of insulin resistance. In oneembodiment, said modulator comprises any one or more substances selectedfrom the group consisting of antisense oligonucleotides, triple helixDNA, ribozymes, RNA aptamer, siRNA and double or single stranded RNAwherein said substances are designed to inhibit gene expression of saidprotein. In yet a further embodiment, said modulator comprises one ormore antibodies to said protein or fragments thereof, wherein saidantibodies or fragments thereof can, e.g., inhibit the biochemicalfunction of said protein.

The invention also pertains to a modulator to a protein selected fromthe group consisting of those disclosed in Table 4 or Table 5 for use asa pharmaceutical. In one embodiment, said modulator comprises any one ormore substances selected from the group consisting of antisenseoligonucleotides, triple helix DNA, ribozymes, RNA aptamer, siRNA anddouble or single stranded RNA wherein said substances are designed toinhibit gene expression of said protein. In yet a further embodiment,said modulator comprises one or more antibodies to said protein orfragments thereof, wherein said antibodies or fragments thereof can,e.g., inhibit the biochemical functions of said protein.

In another aspect, the invention also pertains to a method to identifyproteins involved in the insulin signaling pathway, said methodcomprising providing a transgenic fly whose genome comprises a DNAsequence encoding a polypeptide comprising the dominant negative PI3Kcatalytic subunit Dp110^(D954A), said DNA sequence operably linked to atissue specific expression control sequence, and expressing said DNAsequence, wherein expression of said DNA sequence results in said flydisplaying a transgenic phenotype compared to controls; crossing saidtransgenic fly with a fly containing a mutation in a known or predictedgene; and screening progeny of said crosses for flies that carry saidDNA sequence and said mutation and display modified expression of thetransgenic phenotype as compared to appropriate controls. In oneembodiment, said DNA sequence encodes Dp110^(D954A) and said tissuespecific expression control sequence comprises the eye-specific enhancerey-Gal4 and expression of said DNA sequence results in said flydisplaying the “small eye” phenotype.

In a particular embodiment the invention relates to a method to identifydrug targets for the development of therapeutics to treat, prevent orameliorate pathological conditions associated with dysregulation of theinsulin signaling pathway, including Type II diabetes and Type Asyndrome of insulin resistance, said method comprising identifying thehuman homologs of the Drosophila proteins identified according to themethod discussed above.

Other objects, features, advantages and aspects of the present Inventionwill become apparent to those of skill from the following description.It should be understood, however, that the following description and thespecific examples, while indicating preferred embodiments of theinvention, are given by way of illustration only. Various changes andmodifications within the spirit and scope of the disclosed inventionwill become readily apparent to those skilled in the art from readingthe following description and from reading the other parts of thepresent disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of the eyGal4, UAS, Dp110^(DN) and UASPTEN crossof drosophila transgenes.

FIG. 2 is a schematic of crosses used for screening X chromosome lethalP-element insertion lines.

FIG. 3 is a schematic of crosses used for screening second chromosomelethal P-element insertion lines.

FIG. 4 is a schematic of crosses used for screening third chromosomelethal P-element insertion lines.

DETAILED DESCRIPTION OF THE INVENTION

All patent applications, patents and literature references cited hereinare hereby incorporated by reference in their entirety.

Abbreviations used in the following description include:

IRSs, insulin receptor substrates.

PI3K, phosphoinositide 3-kinase.

PDKs, 3′-phosphoinositide-dependent protein kinases.

PTEN, phosphatase and tensin homolog deleted from chromosome 10.

PKB, protein kinase B, also known as Aktl.

In practicing the present invention, many conventional techniques inmolecular biology, microbiology, and recombinant DNA are used. Thesetechniques are well known and are explained in, for example, CurrentProtocols in Molecular Biology, Volumes I, II, and III, 1997 (F. M.Ausubel ed.); Sambrook et al., 1989, Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; DNA Cloning: A Practical Approach, Volumes 1 and 11, 1985(D. N. Glover ed.); Oligonucleotide Synthesis, 1984 (M. L. Gait ed.);Nucleic Acid Hybridization, 1985, (Hames and Higgins); Transcription andTranslation, 1984 (Hames and Higgins eds.); Animal Cell Culture, 1986(R. I. Freshney ed.); Immobilized Cells and Enzymes, 1986 (IRL Press);Perbal, 1984, A Practical Guide to Molecular Cloning; the series,Methods in Enzymology (Academic Press, Inc.); Gene Transfer Vectors forMammalian Cells, 1987 (J. H. Miller and M. P. Calos eds., Cold SpringHarbor Laboratory); and Methods in Enzymology Vol. 154 and Vol. 155 (Wuand Grossman, and Wu, eds., respectively). Well known Drosophilamolecular genetics techniques can be found, for example, in Robert, D.B., Drosophila, A Practical Approach (IRL Press, Washington D.C. 1986).

Descriptions of flystocks can be found in the Flybase database athttp://flybase.bio.indiana.edu.

Stock centers referred to herein include Bloomington and Szeged stockcenters which are located at Bloomington, Ind. and Szeged, Hungary,respectively.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to “the antibody” is a referenceto one or more antibodies and equivalents thereof known to those skilledin the art, and so forth.

“Nucleic acid sequence”, as used herein, refers to an oligonucleotide,nucleotide or polynucleotide, and fragments or portions thereof, and toDNA or RNA of genomic or synthetic origin that may be single or doublestranded, and represent the sense or antisense strand.

The term “antisense” as used herein, refers to nucleotide sequenceswhich are complementary to a specific DNA or RNA sequence. The term“antisense strand” is used in reference to a nucleic acid strand that iscomplementary to the “sense” strand. Antisense molecules may be producedby any method, including synthesis by ligating the gene(s) of interestin a reverse orientation to a viral promoter which permits the synthesisof a complementary strand. Once introduced into a cell, this transcribedstrand combines natural sequences produced by the cell to form duplexes.These duplexes then block either the further transcription ortranslation. The designation “negative” is sometimes used in referenceto the antisense strand, and “positive” is sometimes used in referenceto the sense strand.

“cDNA” refers to DNA that is complementary to a portion of messenger RNA(mRNA) sequence and is generally synthesized from an mRNA preparationusing reverse transcriptase.

As contemplated herein, antisense oligonucleotides, triple helix DNA,RNA aptamers, ribozymes, siRNA and double or single stranded RNA aredirected to a nucleic acid sequence such that the nucleotide sequencechosen will produce gene-specific inhibition of gene expression. Forexample, knowledge of a nucleotide sequence may be used to design anantisense molecule which gives strongest hybridization to the mRNA.Similarly, ribozymes can be synthesized to recognize specific nucleotidesequences of a gene and cleave it (Cech. J. Amer. Med Assn. 260:3030(1988)). Techniques for the design of such molecules for use in targetedinhibition of gene expression is well known to one of skill in the art.

The individual proteins/polypeptides referred to herein include any andall forms of these proteins including, but not limited to, partialforms, isoforms, variants, precursor forms, the full length protein,fusion proteins containing the sequence or fragments of any of theabove, from human or any other species. Protein homologs or orthologswhich would be apparent to one of skill in the art are included in thisdefinition. It is also contemplated that the term refers to proteinsisolated from naturally occurring sources of any species such as genomicDNA libraries as well as genetically engineered host cells comprisingexpression systems, or produced by chemical synthesis using, forinstance, automated peptide synthesizers or a combination of suchmethods. Means for isolating and preparing such polypeptides are wellunderstood in the art.

The term “sample” as used herein, is used in its broadest sense. Abiological sample from a subject may comprise blood, urine, braintissue, primary cell lines, immortilized cell lines, or other biologicalmaterial with which protein activity or gene expression may be assayed.A biological sample may include, for example, blood, tumors or otherspecimens from which total RNA may be purified for gene expressionprofiling using, for example, conventional glass chip microarraytechnologies such as Affymetrix chips, RT-PCR or other conventionalmethods.

As used herein, the term “antibody” refers to intact molecules as wellas fragments thereof, such as Fa, F(ab′)₂, and Fv, which are capable ofbinding the epitopic determinant. Antibodies that bind specificpolypeptides can be prepared using intact polypeptides or fragmentscontaining small peptides of interest as the immunizing antigen. Thepolypeptides or peptides used to immunize an animal can be derived fromthe translation of RNA or synthesized chemically, and can be conjugatedto a carrier protein. Commonly used carriers that are chemically coupledto peptides include bovine serum albumin and thyroglobulin. The coupledpeptide is then used to immunize an animal (e.g., a mouse, goat,chicken, rat or a rabbit).

The term “humanized antibody” as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

A “therapeutically effective amount” is the amount of drug sufficient totreat, prevent or ameliorate pathological conditions associated withdysregulation of the insulin signaling pathway.

A “transgenic” organism as used herein refers to an organism that hashad extra genetic material inserted into its genome. As used herein, a“transgenic fly” includes embryonic, larval and adult forms ofDrosophila that contain a DNA sequence from the same or another organismrandomly inserted into their genome. Although Drosophila melanogaster ispreferred, it is contemplated that any fly of the genus Drosophila maybe used in the present invention.

As used herein, “ectopic” expression of the transgene refers toexpression of the transgene in a tissue or cell or at a specificdevelopmental stage where it is not normally expressed.

As used herein, “phenotype” refers to the observable physical orbiochemical characteristics of an organism as determined by both geneticmakeup and environmental influences.

The term “transcription factor” refers to any protein required toinititate or regulate transcription in eukaryotes. For example, theeye-specific promoter GMR is a binding site for the eye-specifictranscription factor, GLASS (Moses, K and Rubin, G M Genes Dev.5(4):583-93 (1991)).

“UAS” region as used herein refers to an upstream activating sequencerecognized by the GAL-4 transcriptional activator.

As used herein, a “control” fly refers to a larva or fly that is of thesame genotype as larvae or flies used in the methods of the presentinvention except that the control larva or fly does not carry themutation being tested for modification of phenotype.

As used herein, a “transformation vector” is a modified transposableelement used with the transposable element technique to mediateintegration of a piece of DNA in the genome of the organism and isfamiliar to one of skill in the art.

As used herein, “elevated transcription of mRNA” refers to a greateramount of messenger RNA transcribed from the natural endogenous geneencoding a protein, e.g. a human protein set forth in Table 4 or Table5, compared to control levels. Elevated mRNA levels of a protein, e.g.a. human protein disclosed on Table 4 or Table 5, may be present in atissue or cell of an individual suffering from a pathological conditionassociated with dysregulation of the insulin signaling pathway comparedto levels in a subject not suffering from said condition. In particular,levels in a subject suffering from said condition may be at least abouttwice, preferably at least about five times, more preferably at leastabout ten times, most preferably at least about 100 times the amount ofmRNA found in corresponding tissues in humans who do not suffer fromsaid condition. Such elevated level of mRNA may eventually lead toincreased levels of protein translated from such mRNA in an individualsuffering from a pathological condition associated with dysregulation ofthe insulin signaling pathway as compared to levels in a healthyindividual.

As used herein, a “Drosophila transformation vector” is a DNA plasmidthat contains transposable element sequences and can mediate integrationof a piece of DNA in the genome of the organism. This technology isfamiliar to one of skill in the art.

As used herein, the “small eye phenotype” is characterized by reducedcell size in the eye tissue compared to appropriate controls (Leevers, SJ et al. EMBO J. 1996 Dec. 2; 15 (23):6584-94).

Methods of obtaining transgenic organisms, including transgenicDrosophila, are well known to one skilled in the art. For example, acommonly used reference for P-element mediated transformation isSpradling, 1986, “P element mediated transformation”, In Drosophila: Apractical approach (ed. D. B. Roberts), pp175-197, IRL Press, Oxford,UK. The EP element technology refers to a binary system, utilizing theyeast Gal4 transcriptional activator, that is used to ectopicallyregulate the transcription of endogenous Drosophila genes. Thistechnology is described in Brand and Perrimon, 1993. “Targeted geneexpression as a means of altering cell fates and generating dominantphenotypes”, Development 118, pp401-415 and in: Rorth et al, 1998,“Systematic gain-of-function genetics in Drosophila” Development,125(6), pp1049-1057.

A “host cell,” as used herein, refers to a prokaryotic or eukaryoticcell that contains heterologous DNA that has been introduced into thecell by any means, e.g., electroporation, calcium phosphateprecipitation, microinjection, transformation, viral infection, and thelike.

“Heterologous” as used herein means “of different natural origin” orrepresent a non-natural state. For example, if a host cell istransformed with a DNA or gene derived from another organism,particularly from another species, that gene is heterologous withrespect to that host cell and also with respect to descendants of thehost cell which carry that gene. Similarly, heterologous refers to anucleotide sequence derived from and inserted into the same natural,original cell type, but which is present in a non-natural state, e.g. adifferent copy number, or under the control of different regulatoryelements.

A “vector” molecule is a nucleic acid molecule into which heterologousnucleic acid may be inserted which can then be introduced into anappropriate host cell. Vectors preferably have one or more origin ofreplication, and one or more site into which the recombinant DNA can beinserted. Vectors often have convenient means by which cells withvectors can be selected from those without, e.g., they encode drugresistance genes. Common vectors include plasmids, viral genomes, and(primarily in yeast and bacteria) “artificial chromosomes.”

“Plasmids” generally are designated herein by a lower case p precededand/or followed by capital letters and/or numbers, in accordance withstandard naming conventions that are familiar to those of skill in theart. Starting plasmids disclosed herein are either commerciallyavailable, publicly available on an unrestricted basis, or can beconstructed from available plasmids by routine application of wellknown, published procedures. Many plasmids and other cloning andexpression vectors that can be used in accordance with the presentinvention are well known and readily available to those of skill in theart. Moreover, those of skill readily may construct any number of otherplasmids suitable for use in the invention. The properties, constructionand use of such plasmids, as well as other vectors, in the presentinvention will be readily apparent to those of skill from the presentdisclosure.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated, even ifsubsequently reintroduced into the natural system. Such polynucleotidescould be part of a vector and/or such polynucleotides or polypeptidescould be part of a composition, and still be isolated in that suchvector or composition is not part of its natural environment.

As used herein, the terms “transcriptional control sequence” or“expression control sequence” refer to DNA sequences, such as initiatorsequences, enhancer sequences, and promoter sequences, which induce,repress, or otherwise control the transcription of a protein encodingnucleic acid sequences to which they are operably linked. They may betissue specific and developmental-stage specific. A “humantranscriptional control sequence” is a transcriptional control sequencenormally found associated with the human gene encoding a polypeptide setforth in Table 4 of the present invention as it is found in therespective human chromosome. A “non-human transcriptional controlsequence” is any transcriptional control sequence not found in the humangenome.

The term “polypeptide” is used interchangeably herein with the terms“polypeptides” and “protein(s)”.

A “chemical derivative” of a protein set forth in Table 4 or Table 5 ofthe invention is a polypeptide that contains additional chemicalmoieties not normally a part of the molecule. Such moieties may improvethe molecule's solubility, absorption, biological half life, etc. Themoieties may alternatively decrease the toxicity of the molecule,eliminate or attenuate any undesirable side effect of the molecule, etc.Moieties capable of mediating such effects are disclosed, for example,in Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co.,Easton, Pa. (1980).

The ability of a substance to “modulate” a protein set forth in Table 4or Table 5 (i.e. “a modulator of a protein selected from the groupconsisting of those disclosed in Table 4 or Table 5”) includes, but isnot limited to, the ability of a substance to inhibit the activity ofsaid protein and/or inhibit the gene expression of said protein. Suchmodulation could also involve effecting the ability of other proteins tointeract with said protein, for example related regulatory proteins orproteins that are modified by said protein.

The term “agonist”, as used herein, refers to a molecule (i.e.modulator) which, directly or indirectly, may modulate a polypeptide(e.g. a polypeptide set forth in Table 4 or Table 5) and which increasethe biological activity of said polypeptide. Agonists may includeproteins, nucleic acids, carbohydrates, or other molecules A modulatorthat enhances gene transcription or the biochemical function of aprotein is something that increases transcription or stimulates thebiochemical properties or activity of said protein, respectively.

The terms “antagonist” or “inhibitor” as used herein, refer to amolecule (i.e. modulator) which directly or indirectly may modulate apolypeptide (e.g. a polypeptide set forth in Table 4 or Table 5) whichblocks or inhibits the biological activity of said polypeptide.Antagonists and inhibitors may include proteins, nucleic acids,carbohydrates, or other molecules. A modulator that inhibits geneexpression or the biochemical function of a protein is something thatreduces gene expression or biological activity of said protein,respectively.

As used herein, “pathological condition associated with dysregulation ofthe insulin signaling pathway” includes, but is not limited to,diabetes, e.g., Type II diabetes, gestational diabetes and the Type Asyndrome of insulin resistance.

As generally referred to herein, a protein or gene selected from thegroup consisting of those disclosed in Table 4 or Table 5 refers to thehuman form of the gene.

“In vivo models of a pathological condition associated withdysregulation of the insulin signaling pathway” include those in vivomodels of diabetes familiar to those of skill in the art. Such in vivomodels include: an association of the common pro12 allele in PPAR-gammawith type-2 diabetes (Altshuler, D. et al., Nature Genet. 76-80, 2000),defects in the human insulin receptor gene (Kadowaki, T., et al. Science240: 787-790, 1988), defects in the insulin receptor substrate 1 gene inmouse (Abe, H.; et al., J. Clin. Invest. 101: 1784-1788, 1998) anddefects in glycogen sythase in humans (Groop, L. C et al., New Eng. J.Med. 328: 10-14, 1993).

The present invention discloses a method comprising using as a modelorganism a transgenic fly, Drosophila melanogaster, whose genomecomprises a DNA sequence encoding the gene Dp110^(D954A) Conventionalexpression control systems may be used to achieve ectopic expression ofproteins of interest, including the Dp110^(D954A) peptide. Suchexpression may result in the disturbance of biochemical pathways and thegeneration of altered phenotypes. One such expression control systeminvolves direct fusion of the DNA sequence to expression controlsequences of tissue-specifically expressed genes, such as promoters orenhancers. A tissue specific expression control system that may be usedis the binary Gal4-transcriptional activation system (Brand andPerrimon, Development 118:401-415 (1993)).

The Gal4 system uses the yeast transcriptional activator Gal4, to drivethe expression of a gene of interest in a tissue specific manner. TheGal4 gene has been randomly inserted into the fly genome, using aconventional transformation system, so that it has come under thecontrol of genomic enhancers that drive expression in a temporal andtissue-specific manner. Individual strains of flies have beenestablished, called “drivers”, that carry those insertions (Brand andPerrimon, Development 118:401-415 (1993)).

In the Gal4 system, a gene of interest is cloned into a transformationvector, so that its transcription is under the control of the UASsequence (Upstream Activating Sequence), the Gal4-responsive element.When a fly strain that carries the UAS-gene of interest sequence iscrossed to a fly strain that expresses the Gal4 gene under the controlof a tissue specific enhancer, the gene will be expressed in a tissuespecific pattern.

In order to generate phenotypes that are easily visible in adult tissuesand can thus be used in genetic screens, Gal4 “drivers” that driveexpression in later stages of the fly development may be used in thepresent invention. Using these drivers, expression would result inpossible defects in the wings, the eyes, the legs, different sensoryorgans and the brain. These “drivers” include, for example,apterous-Gal4 (wings), elav-Gal4 (CNS), sevenless-Gal4, eyeless-Gal4(also called ey-Gal4) and pGMR-Gal4 (eyes). Descriptions of the Gal4lines and notes about their specific expression patterns is available inFlybase (http://flybase.bio.indiana.edu)

Various DNA constructs may be used to generate the transgenic Drosophilamelanogaster disclosed herein. For example, the construct may containthe Dp110^(D954A) sequence cloned into the pUAST vector (Brand andPerrimon, Development 118:401-415 (1993)) which places the UAS sequenceupstream of the transcribed region. Insertion of these constructs intothe fly genome may occur through P-element recombination, Hobo elementrecombination (Blackman et al., EMBO J. 8:211-217 (1989)), homologousrecombination (Rong and Golic, Science 288:2013-2018 (2000)) or otherstandard techniques known to one of skill in the art.

As discussed above, an ectopically expressed gene may result in analtered phenotype by disruption of a particular biochemical pathway.Mutations in genes acting in the same biochemical pathway are expectedto cause modification of the altered phenotype. Thus, for example, atransgenic fly carrying both eyeless-Gal4 and UAS-Dp110^(D954A) can beused to identify genes acting in the insulin signalling pathway bycrossing this transgenic fly with a fly containing a mutation in a knownor predicted gene; and screening progeny of the crosses for flies thatdisplay quantitative or qualitative modification of the alteredphenotype of the eyeless-Gal4/Dp110^(D954A) transgenic fly, as comparedto controls. Thus, this system is extremely beneficial for theelucidation of the function of Dp110^(D954A) products, as well as theidentification of other genes that directly or indirectly interact withthem. Mutations that can be screened include, but are not limited to,loss-of-function alleles of known genes, deletion strains,“enhancer-trap” strains generated by the P-element and gain-of-functionmutations generated by random insertions into the Drosophila genome of aGal4-inducible construct that can activate the ectopic expression ofgenes in the vicinity of its insertion. It is contemplated herein thatgenes involved in the insulin signaling pathway can be identified inthis manner and these genes can then serve as targets for thedevelopment of therapeutics to treat pathological conditions associatedwith dysregulation in the insulin signaling pathway.

Nucleic acid molecules of the human homologs of the target polypeptidesidentifed according to the methods of the present invention anddisclosed herein may act as target gene antisense molecules, useful, forexample, in target gene regulation and/or as antisense primers inamplification reactions of target gene nucleic acid sequences. Further,such sequences may be used as part of ribozyme and/or triple helixsequences or as targets for siRNA or double or single stranded RNA,which may be employed for gene regulation. Still further, such moleculesmay be used as components of diagnostic kits as disclosed herein.

In cases where the gene identified using the methods of the presentinvention is the normal, or wild type, gene, this gene may be used toisolate mutant alleles of the gene. Such isolation is preferable inprocesses and disorders which are known or suspected to have a geneticbasis. Mutant alleles may be isolated from individuals either known orsuspected to have a genotype which contributes to disease symptomsrelated to pathological conditions associated with dysregulation of theinsulin signaling pathway, including, but not limited to, conditionssuch as Type II diabetes or the Type A syndrome of insulin resistance.(Taylor, S. I and Ariogluo, E. (1998) J. Basic Clin. Physiol. Pharmacol.9, 419-439). Mutant alleles and mutant allele products may then beutilized in the diagnostic assay systems described herein.

A cDNA of the mutant gene may be isolated, for example, by using PCR, atechnique which is well known to those of skill in the art. In thiscase, the first cDNA strand may be synthesized by hybridizing anoligo-dT oligonucleotide to mRNA isolated from tissue known or suspectedto be expressed in an individual putatively carrying the mutant allele,and by extending the new strand with reverse transcriptase. The secondstrand of the cDNA is then synthesized using an oligonucleotide thathybridizes specifically to the 5′ end of the normal gene. Using thesetwo primers, the product is then amplified via PCR, cloned into asuitable vector, and subjected to DNA sequence analysis through methodswell known to those of skill in the art. By comparing the DNA sequenceof the mutant gene to that of the normal gene, the mutation(s)responsible for the loss or alteration of function of the mutant geneproduct can be ascertained.

Alternatively, a genomic or cDNA library can be constructed and screenedusing DNA or RNA, respectively, from a tissue known to or suspected ofexpressing the gene of interest in an individual suspected of or knownto carry the mutant allele. The normal gene or any suitable fragmentthereof may then be labeled and used as a probe to identify thecorresponding mutant allele in the library. The clone containing thisgene may then be purified through methods routinely practiced in theart, and subjected to sequence analysis as described above.

Additionally, an expression library can be constructed utilizing DNAisolated from or cDNA synthesized from a tissue known to or suspected ofexpressing the gene of interest in an individual suspected of or knownto carry the mutant allele. In this manner, gene products made by theputatively mutant tissue may be expressed and screened using standardantibody screening techniques in conjunction with antibodies raisedagainst the normal gene product, as described below. (For screeningtechniques, see, for example, Harlow, E. and Lane, eds., 1988,“Antibodies: A Laboratory Manual”, Cold Spring Harbor Press, Cold SpringHarbor). In cases where the mutation results in an expressed geneproduct with altered function (e.g., as a result of a missensemutation), a polyclonal set of antibodies are likely to cross-react withthe mutant gene product. Library clones detected via their reaction withsuch labeled antibodies can be purified and subjected to sequenceanalysis as described above.

In another embodiment, nucleic acids comprising a sequence encoding apolypeptide set forth in Table 4, or Table 5 or functional derivativesthereof, may be administered to promote normal biological function, forexample, normal insulin mediated signal transduction, by way of genetherapy. Gene therapy refers to therapy performed by the administrationof a nucleic acid to a subject. In this embodiment of the invention, thenucleic acid produces its encoded protein that mediates a therapeuticeffect by promoting a normal insulin signaling pathway.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

In a preferred aspect, the therapeutic comprises a nucleic acid for aTable 4 or Table 5 polypeptide that is part of an expression vector thatexpresses a Table 4 or Table 5 protein or fragment or chimeric proteinthereof in a suitable host. In particular, such a nucleic acid has apromoter operably linked to the Table 4 or Table 5 protein codingregion, said promoter being inducible or constitutive, and, optionally,tissue-specific. In another particular embodiment, a nucleic acidmolecule is used in which the Table 4 or Table 5 protein codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the Table 4 or Table 5nucleic acid (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA86:8932-8935; Zijistra et al., 1989, Nature 342:435-438).

Delivery of the nucleic acid into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vector, or indirect, in which case, cells arefirst transformed with the nucleic acid in vitro, then transplanted intothe patient. These two approaches are known, respectively, as in vivo orex vivo gene therapy.

In a specific embodiment, the nucleic acid is directly administered invivo, where it is expressed to produce the encoded product. This can beaccomplished by any of numerous methods known in the art, e.g., byconstructing it as part of an appropriate nucleic acid expression vectorand administering it so that it becomes intracellular, e.g., byinfection using a defective or attenuated retroviral or other viralvector (see, e.g., U.S. Pat. No. 4,980,286 and others mentioned infra),or by direct injection of naked DNA, or by use of microparticlebombardment (e.g., a gene gun; Biolistic, Dupont), or coating withlipids or cell-surface receptors or transfecting agents, encapsulationin liposomes, microparticles, or microcapsules, or by administering itin linkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see e.g., U.S. Pat. Nos. 5,166,320; 5,728,399; 5,874,297;and 6,030,954, all of which are incorporated by reference herein intheir entirety) (which can be used to target cell types specificallyexpressing the receptors), etc. In another embodiment, a nucleicacid-ligand complex can be formed in which the ligand comprises afusogenic viral peptide to disrupt endosomes, allowing the nucleic acidto avoid lysosomal degradation. In yet another embodiment, the nucleicacid can be targeted in vivo for cell specific uptake and expression, bytargeting a specific receptor (see, e.g., PCT Publications WO 92/06180;WO 92/22635; WO92/20316; WO93/14188; and WO 93/20221). Alternatively,the nucleic acid can be introduced intracellularly and incorporatedwithin host cell DNA for expression, by homologous recombination (see,e.g., U.S. Pat. Nos. 5,413,923; 5,416,260; and 5,574,205; and Zijistraet al., 1989, Nature 342:435-438).

In a specific embodiment, a viral vector that contains a nucleic acidencoding a Table 4 or Table 5 polypeptide is used. For example, aretroviral vector can be used (see, e.g., U.S. Pat. Nos. 5,219,740;5,604,090; and 5,834,182). These retroviral vectors have been modifiedto delete retroviral sequences that are not necessary for packaging ofthe viral genome and integration into host cell DNA. The nucleic acidfor the Table 4 or Table 5 polypeptide to be used in gene therapy iscloned into the vector, which facilitates delivery of the gene into apatient.

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Methods for conductingadenovirus-based gene therapy are described in, e.g., U.S. Pat. Nos.5,824,544; 5,868,040; 5,871,722; 5,880,102; 5,882,877; 5,885,808;5,932,210; 5,981,225; 5,994,106; 5,994,132; 5,994,134; 6,001,557; and6,033,8843, all of which are incorporated by reference herein in theirentirety.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy. Methods for producing and utilizing AAV are described, e.g., inU.S. Pat. Nos. 5,173,414; 5,252,479; 5,552,311; 5,658,785; 5,763,416;5,773,289; 5,843,742; 5,869,040; 5,942,496; and 5,948,675, all of whichare incorporated by reference herein in their entirety.

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. In a preferred embodiment, epithelial cellsare injected, e.g., subcutaneously. In another embodiment, recombinantskin cells may be applied as a skin graft onto the patient. Recombinantblood cells (e.g., hematopoietic stem or progenitor cells) arepreferably administered intravenously. The amount of cells envisionedfor use depends on the desired effect, patient state, etc., and can bedetermined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the patient.

In an embodiment in which recombinant cells are used in gene therapy,the nucleic acid of a polypeptide set forth in Table 4 or Table 5 isintroduced into the cells such that it is expressible by the cells ortheir progeny, and the recombinant cells are then administered in vivofor therapeutic effect. In a specific embodiment, stem or progenitorcells are used. Any stem-and/or progenitor cells that can be isolatedand maintained in vitro can potentially be used in accordance with thisembodiment of the present invention. Such stem cells include but are notlimited to hematopoietic stem cells (HSC), stem cells of epithelialtissues such as the skin and the lining of the gut, embryonic heartmuscle cells, liver stem cells (see, e.g., WO 94/08598), and neural stemcells (Stemple and Anderson, 1992, Cell 71:973-985).

Epithelial stem cells (ESCs) or keratinocytes can be obtained fromtissues such as the skin and the lining of the gut by known procedures(Rheinwald, 1980, Meth. Cell Bio. 21A:229). In stratified epithelialtissue such as the skin, renewal occurs by mitosis of stem cells withinthe germinal layer, the layer closest to the basal lamina. Stem cellswithin the lining of the gut provide for a rapid renewal rate of thistissue. ESCs or keratinocytes obtained from the skin or lining of thegut of a patient or donor can be grown in tissue culture (Pittelkow andScott, 1986, Mayo Clinic Proc. 61:771). If the ESCs are provided by adonor, a method for suppression of host versus graft reactivity (e.g.,irradiation, drug or antibody administration to promote moderateimmunosuppression) can also be used.

With respect to hematopoietic stem cells (HSC), any technique whichprovides for the isolation, propagation, and maintenance in vitro of HSCcan be used in this embodiment of the invention. Techniques by whichthis may be accomplished include (a) the isolation and establishment ofHSC cultures from bone marrow cells isolated from the future host, or adonor, or (b) the use of previously established long-term HSC cultures,which may be allogeneic or xenogeneic. Non-autologous HSC are usedpreferably in conjunction with a method of suppressing transplantationimmune reactions of the future host/patient. In a particular embodimentof the present invention, human bone marrow cells can be obtained fromthe posterior iliac crest by needle aspiration (see, e.g., Kodo et al.,1984, J. Clin. Invest. 73:1377-1384). In a preferred embodiment of thepresent invention, the HSCs can be made highly enriched or insubstantially pure form. This enrichment can be accomplished before,during, or after long-term culturing, and can be done by any techniquesknown in the art. Long-term cultures of bone marrow cells can beestablished and maintained by using, for example, modified Dexter cellculture techniques (Dexter et al., 1977, J. Cell Physiol. 91:335) orWitlock-Witte culture techniques (Witlock and Witte, 1982, Proc. Natl.Acad. Sci. USA 79:3608-3612).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

A further embodiment of the present invention relates to a method totreat, prevent or ameliorate a pathological condition associated withdysregulation of the insulin signaling pathway that comprisesadminstering to a subject in need thereof an effective amount of amodulator of a protein selected from the group consisting of thosedisclosed in Table 4 or Table 5. In one embodiment, the modulatorcomprises one or more antibodies to said protein, or fragments thereof,wherein said antibodies or fragments thereof can inhibit the biochemicalfunction of said protein in said subject

Described herein are methods for the production of antibodies capable ofspecifically recognizing one or more differentially expressed geneepitopes. Such antibodies may include, but are not limited to polyclonalantibodies, monoclonal antibodies (mAbs), humanized or chimericantibodies, single chain antibodies, Fab fragments, F(ab′)₂ fragments,fragments produced by a Fab expression library, anti-idiotypic (anti-id)antibodies, and epitope-binding fragments of any of the above. Suchantibodies may be used, for example, in the detection of a targetprotein in a biological sample, or alternatively, as a method for theinhibition of the biochemical function of the protein. Thus, suchantibodies may be utilized as part of disease treatment methods, and/ormay be used as part of diagnostic techniques whereby patients may betested e.g., for abnormal levels of polypeptides set forth in Table 4 orTable 5, or for the presence of abnormal forms of these polypeptides.

For the production of antibodies to the Table 4 or Table 5 polypeptides,various host animals may be immunized by injection with thesepolypeptides, or a portion thereof. Such host animals may include butare not limited to rabbits, mice, goats, chickens and rats, to name buta few. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and Corynebacterium parvum.

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen,such as target gene product, or an antigenic functional derivativethereof. For the production of polyclonal antibodies, host animals suchas those described above, may be immunized by injection with a Table 4or Table 5 polypeptide, or a portion thereof, supplemented withadjuvants as also described above.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, may be obtained by any technique that providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to the hybridoma techniqueof Kohler and Milstein, (1975, Nature 256:495-497; and U.S. Pat. No.4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may be of any immunoglobulin class includingIgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridomaproducing the mAb of this invention may be cultivated in vitro or invivo. Production of high titers of mAbs in vivo makes this the presentlypreferred method of production.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.,81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableor hypervariable region derived from a murine mAb and a humanimmunoglobulin constant region.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423-426;Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Wardet al., 1989, Nature 334:544-546) can be adapted to producedifferentially expressed gene-single chain antibodies. Single chainantibodies are formed by linking the heavy and light chain fragments ofthe Fv region via an amino acid bridge, resulting in a single chainpolypeptide.

Most preferably, techniques useful for the production of “humanizedantibodies” can be adapted to produce antibodies to the polypeptides,fragments, derivatives, and functional equivalents disclosed herein.Such techniques are disclosed in U.S. Pat. Nos. 5,932,448; 5,693,762;5,693,761; 5,585,089; 5,530,101; 5,910,771; 5,569,825; 5,625,126;5,633,425; 5,789,650; 5,545,580; 5,661,016; and 5,770,429, thedisclosures of all of which are incorporated by reference herein intheir entirety.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, such fragments include but are notlimited to: the F(ab′)₂ fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed (Huse et al.,1989, Science, 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

As contemplated herein, an antibody of the present invention can bepreferably used in a diagnostic kit for detecting levels of a proteindisclosed in Table 4 or Table 5 in a biological sample as well as in amethod to diagnose subjects suffering from pathological conditionsassociated with dysregulation of the insulin signaling pathway who maybe suitable candidates for treatment with moauiators to a proteinselected from the group consisting of those disclosed in Table 4 orTable 5. Preferably, said detecting step comprises contacting saidappropriate tissue cell (e.g. biological sample) with an antibody whichspecifically binds to a Table 4 or Table 5 polypeptide or a fragmentthereof and detecting specific binding of said antibody with apolypeptide in said appropriate tissue, cell or sample wherein detectionof specific binding to a polypeptide indicates the presence of apolypeptide set forth in Table 4, or Table 5 or a fragment thereof.

Particularly preferred, for ease of detection, is the sandwich assay, ofwhich a number of variations exist, all of which are intended to beencompassed by the present invention. For example, in a typical forwardassay, unlabeled antibody is immobilized on a solid substrate and thesample to be tested brought into contact with the bound molecule. Aftera suitable period of incubation, for a period of time sufficient toallow formation of an antibody-antigen binary complex. At this point, asecond antibody, labeled with a reporter molecule capable of inducing adetectable signal, is then added and incubated, allowing time sufficientfor the formation of a ternary complex of antibody-antigen-labeledantibody. Any unreacted material is washed away, and the presence of theantigen is determined by observation of a signal, or may be quantitatedby comparing with a control sample containing known amounts of antigen.Variations on the forward assay include the simultaneous assay, in whichboth sample and antibody are added simultaneously to the bound antibody,or a reverse assay in which the labeled antibody and sample to be testedare first combined, incubated and added to the unlabeled surface boundantibody. These techniques are well known to those skilled in the art,and the possibility of minor variations will be readily apparent. Asused herein, “sandwich assay” is intended to encompass all variations onthe basic two-site technique. For the immunoassays of the presentinvention, the only limiting factor is that the labeled antibody be anantibody which is specific for a Table 4 or Table 5 polypeptide or afragment thereof.

The most commonly used reporter molecules in this type of assay areeither enzymes, fluorophore- or radionuclide-containing molecules. Inthe case of an enzyme immunoassay, an enzyme is conjugated to the secondantibody, usually by means of glutaraldehyde or periodate. As will bereadily recognized, however, a wide variety of different ligationtechniques exist, which are well-known to the skilled artisan. Commonlyused enzymes include horseradish peroxidase, glucose oxidase,beta-galactosidase and alkaline phosphatase, among others. Thesubstrates to be used with the specific enzymes are generally chosen forthe production, upon hydrolysis by the corresponding enzyme, of adetectable color change. For example, p-nitrophenyl phosphate issuitable for use with alkaline phosphatase conjugates; for peroxidaseconjugates, 1,2-phenylenediamine or toluidine are commonly used. It isalso possible to employ fluorogenic substrates, which yield afluorescent product rather than the chromogenic substrates noted above.A solution containing the appropriate substrate is then added to thetertiary complex. The substrate reacts with the enzyme linked to thesecond antibody, giving a qualitative visual signal, which may befurther quantitated, usually spectrophotometrically, to give anevaluation of the amount of Table 4 or Table 5 polypeptide which ispresent in the serum sample.

Alternately, fluorescent compounds, such as fluorescein and rhodamine,may be chemically coupled to antibodies without altering their bindingcapacity. When activated by illumination with light of a particularwavelength, the fluorochrome-labeled antibody absorbs the light energy,inducing a state of excitability in the molecule, followed by emissionof the light at a characteristic longer wavelength. The emission appearsas a characteristic color visually detectable with a light microscope.Immunofluorescence and EIA techniques are both very well established inthe art and are particularly preferred for the present method. However,other reporter molecules, such as radioisotopes, chemiluminescent orbioluminescent molecules may also be employed. It will be readilyapparent to the skilled artisan how to vary the procedure to suit therequired use.

The pharmaceutical compositions of the present invention may alsocomprise substances that inhibit the expression of a protein disclosedin Table 4 or Table 5 at the nucleic acid level. Such molecules includeribozymes, antisense oligonucleotides, triple helix DNA, RNA aptamers,siRNA and/or double or single stranded RNA directed to an appropriatenucleotide sequence of nucleic acid encoding such a protein. Theseinhibitory molecules may be created using conventional techniques by oneof skill in the art without undue burden or experimentation. Forexample, modifications (e.g. inhibition) of gene expression can beobtained by designing antisense molecules, DNA or RNA, to the controlregions of the genes encoding the polypeptides discussed herein, i.e. topromoters, enhancers, and introns. For example, oligonucleotides derivedfrom the transcription initiation site, e.g., between positions −10 and+10 from the start site may be used. Notwithstanding, all regions of thegene may be used to design an antisense molecule in order to createthose which gives strongest hybridization to the mRNA and such suitableantisense oligonucleotides may be produced and identified by standardassay procedures familiar to one of skill in the art.

Similarly, inhibition of gene expression may be achieved using “triplehelix” base-pairing methodology. Triple helix pairing is useful becauseit causes inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature (Gee, J. E. et al. (1994) In: Huber, B.E. and B. I. Carr, Molecular and Immunologic Approaches, FuturaPublishing Co., Mt. Kisco, N.Y.). These molecules may also be designedto block translation of mRNA by preventing the transcript from bindingto ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to inhibit geneexpression by catalyzing the specific cleavage of RNA. The mechanism ofribozyme action involves sequence-specific hybridization of the ribozymemolecule to complementary target RNA, followed by endonucleolyticcleavage. Examples which may be used include engineered “hammerhead” or“hairpin” motif ribozyme molecules that can be designed to specificallyand efficiently catalyze endonucleolytic cleavage of gene sequences.Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Ribozyme methods include exposing a cell to ribozymes or inducingexpression in a cell of such small RNA ribozyme molecules (Grassi andMarini, 1996, Annals of Medicine 28: 499-510; Gibson, 1996, Cancer andMetastasis Reviews 15: 287-299). Intracellular expression of hammerheadand hairpin ribozymes targeted to mRNA corresponding to at least one ofthe genes discussed herein can be utilized to inhibit protein encoded bythe gene.

Ribozymes can either be delivered directly to cells, in the form of RNAoligonucleotides incorporating ribozyme sequences, or introduced intothe cell as an expression vector encoding the desired ribozymal RNA.Ribozymes can be routinely expressed in vivo in sufficient number to becatalytically effective in cleaving mRNA, and thereby modifying mRNAabundance in a cell (Cotten et al., 1989 EMBO J. 8:3861-3866). Inparticular, a ribozyme coding DNA sequence, designed according toconventional, well known rules and synthesized, for example, by standardphosphoramidite chemistry, can be ligated into a restriction enzyme sitein the anticodon stem and loop of a gene encoding a tRNA, which can thenbe transformed into and expressed in a cell of interest by methodsroutine in the art. Preferably, an inducible promoter (e.g., aglucocorticoid or a tetracycline response element) is also introducedinto this construct so that ribozyme expression can be selectivelycontrolled. For saturating use, a highly and constituently activepromoter can be used. tDNA genes (i.e., genes encoding tRNAs) are usefulin this application because of their small size, high rate oftranscription, and ubiquitous expression in different kinds of tissues.

Therefore, ribozymes can be routinely designed to cleave virtually anymRNA sequence, and a cell can be routinely transformed with DNA codingfor such ribozyme sequences such that a controllable and catalyticallyeffective amount of the ribozyme is expressed. Accordingly, theabundance of virtually any RNA species in a cell can be modified orperturbed.

Ribozyme sequences can be modified in essentially the same manner asdescribed for antisense nucleotides, e.g., the ribozyme sequence cancomprise a modified base moiety.

RNA aptamers can also be introduced into or expressed in a cell tomodify RNA abundance or activity. RNA aptamers are specific RNA ligandsfor proteins, such as for Tat and Rev RNA (Good et al., 1997, GeneTherapy 4: 45-54) that can specifically inhibit their translation.

Gene specific inhibition of gene expression may also be achieved usingconventional double or single stranded RNA technologies. A descriptionof such technology may be found in WO 99/32619 which is herebyincorporated by reference in its entirety. In addition, siRNA technologyhas also proven useful as a means to inhibit gene expression (Cullen, BRNat. Immunol. 2002 July;3(7):597-9, Martinez, J. et al. Cell 2002September.6;110(5):563).

Antisense molecules, triple helix DNA, RNA aptamers, dsRNA, ssRNA, siRNAand ribozymes of the present invention may be prepared by any methodknown in the art for the synthesis of nucleic acid molecules. Theseinclude techniques for chemically synthesizing oligonucleotides such assolid phase phosphoramidite chemical synthesis. Alternatively, RNAmolecules may be generated by in vitro and in vivo transcription of DNAsequences encoding the genes of the polypeptides discussed herein. SuchDNA sequences may be incorporated into a wide variety of vectors withsuitable RNA polymerase promoters such as T7 or SP6. Alternatively, cDNAconstructs that synthesize antisense RNA constitutively or inducibly canbe introduced into cell lines, cells, or tissues.

Vectors may be introduced into cells or tissues by many available means,and may be used in vivo, in vitro or ex vivo. For ex vivo therapy,vectors may be introduced into stem cells taken from the patient andclonally propagated for autologous transplant back into that samepatient. Delivery by transfection and by liposome injections may beachieved using methods that are well known in the art.

Detection of mRNA levels of proteins disclosed herein may comprisecontacting a biological sample or even contacting an isolated RNA or DNAmolecule derived from a biological sample with an isolated nucleotidesequence of at least about 20 nucleotides in length that hybridizesunder high stringency conditions (e.g. 0.1×SSPE or SSC, 0.1% SDS, 65°C.) with the isolated nucleotide sequence encoding a polypeptide setforth in Table 4 or Table 5. Hybridization conditions may be highlystringent or less highly stringent. In instances wherein the nucleicacid molecules are deoxyoligonucleotides (“oligos”), highly stringentconditions may refer, e.g., to washing in 6×SSC/0.05% sodiumpyrophosphate at 37° C. (for 14-base oligos), 48° C. (for 17-baseoligos), 55° C. (for 20-base oligos), and 60° C. (for 23-base oligos).Suitable ranges of such stringency conditions for nucleic acids ofvarying compositions are described in Krause and Aaronson (1991),Methods in Enzymology, 200:546-556 in addition to Maniatis et al., citedabove.

In some cases, detection of a mutated form of the gene which isassociated with a dysfunction will provide a diagnostic tool that canadd to, or define, a diagnosis of a disease, or susceptibility to adisease, which results from under-expression, over-expression or alteredspatial or temporal expression of the gene. Individuals carryingmutations in the gene may be detected at the DNA level by a variety oftechniques.

Nucleic acids, in particular mRNA, for diagnosis may be obtained from asubject's cells, such as from blood, urine, saliva, tissue biopsy orautopsy material. The genomic DNA may be used directly for detection ormay be amplified enzymatically by using PCR or other amplificationtechniques prior to analysis. RNA or cDNA may also be used in similarfashion. Deletions and insertions can be detected by a change in size ofthe amplified product in comparison to the normal genotype. Pointmutations can be identified by hybridizing amplified DNA to labelednucleotide sequences encoding a polypeptide encoded by a gene disclosedin Table 4 or Table 5. Perfectly matched sequences can be distinguishedfrom mismatched duplexes by RNase digestion or by differences in meltingtemperatures. DNA sequence differences may also be detected byalterations in electrophoretic mobility of DNA fragments in gels, withor without denaturing agents, or by direct DNA sequencing (e.g., Myerset al., Science (1985) 230:1242). Sequence changes at specific locationsmay also be revealed by nuclease protection assays, such as RNase and S1protection or the chemical cleavage method (see Cotton et al., Proc NatlAcad Sci USA (1985) 85: 4397-4401). In addition, an array ofoligonucleotides probes comprising nucleotide sequence encoding theTable 4 or Table 5 polypeptides or fragments of such nucleotidesequences can be constructed to conduct efficient screening of e.g.,genetic mutations. Array technology methods are well known and havegeneral applicability and can be used to address a variety of questionsin molecular genetics including gene expression, genetic linkage, andgenetic variability (see for example: M. Chee et al., Science, Vol 274,pp 610-613 (1996)).

The diagnostic assays offer a process for diagnosing or determining asusceptibility to disease through detection of mutation in the gene of apolypeptide set forth in Table 4 or Table 5 by the methods described. Inaddition, such diseases may be diagnosed by methods comprisingdetermining from a sample derived from a subject an abnormally decreasedor increased level of polypeptide or mRNA. Decreased or increasedexpression can be measured at the RNA level using any of the methodswell known in the art for the quantitation of polynucleotides, such as,for example, nucleic acid amplification, for instance PCR, RT-PCR, RNaseprotection, Northern blotting and other hybridization methods. Assaytechniques that can be used to determine levels of a protein, such as apolypeptide of the present invention, in a sample derived from a hostare well-known to those of skill in the art. Such assay methods includeradioimmunoassays, competitive-binding assays, Western Blot analysis andELISA assays.

Thus in another aspect, the present invention relates to a diagnostickit for detecting mRNA levels (or protein levels) which comprises:

-   (a) a polynucleotide of a polypeptide set forth in Table 4, Table 5    or a fragment thereof;-   (b) a nucleotide sequence complementary to that of (a);-   (c) a polypeptide of Table 4 or Table 5 of the present invention    encoded by the polynucleotide of (a),-   (d) an antibody to the polypeptide of (c)-   (e) an RNAi sequence complementary to that of (a)    It will be appreciated that in any such kit, (a), (b), (c), (d)    or (e) may comprise a substantial component. Such a kit will be of    use in diagnosing a disease or susceptibility to a disease,    particularly to a disease or condition associated with dysregulation    of the insulin signaling pathway, for example, Type II diabetes or    the Type A syndrome of insulin resistance.

The nucleotide sequences of the present invention are also valuable forchromosome localization. The sequence is specifically targeted to, andcan hybridize with, a particular location on an individual humanchromosome. The mapping of relevant sequences to chromosomes is animportant first step in correlating those sequences with gene associateddisease. Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. Such data are found in, for example,V. McKusick, Mendelian Inheritance in Man (available on-line throughJohns Hopkins University Welch Medical Library). The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion are then identified through linkage analysis (coinheritance ofphysically adjacent genes).

The differences in the cDNA or genomic sequence between affected andunaffected individuals can also be determined. If a mutation is observedin some or all of the affected individuals but not in any normalindividuals, then the mutation is likely to be the causative agent ofthe disease.

An additional embodiment of the invention relates to the administrationof a pharmaceutical composition, in conjunction with a pharmaceuticallyacceptable carrier, excipient or diluent, for any of the therapeuticeffects discussed above. Such pharmaceutical compositions may comprise,for example, a polypeptide set forth in Table 4 or Table 5, antibodiesto that polypeptide, mimetics, agonists, antagonists, inhibitors orother modulators of function of a Table 4 or Table 5 polypeptide or genetherefore. The compositions may be administered alone or in combinationwith at least one other agent, such as stabilizing compound, which maybe administered in any sterile, biocompatible pharmaceutical carrier,including, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs or hormones.

In addition, any of the therapeutic proteins, antagonists, antibodies,agonists, antisense sequences or other modulators described above may beadministered in combination with other appropriate therapeutic agents.Selection of the appropriate agents for use in combination therapy maybe made by one of ordinary skill in the art, according to conventionalpharmaceutical principles. The combination of therapeutic agents may actsynergistically to effect the treatment, prevention or amelioration ofpathological conditions associated with abnormalities in the insulinsignaling pathway. Using this approach, one may be able to achievetherapeutic efficacy with lower dosages of each agent, thus reducing thepotential for adverse side effects. Antagonists, agonists and othermodulators of the human polypeptides set forth in Table 4 or Table 5 andgenes encoding said polypeptides may be made using methods which aregenerally known in the art.

The pharmaceutical compositions encompassed by the invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-articular, intra-arterial,intramedullary, intrathecal, intraventricular, transdermal,subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual,or rectal means.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated m aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances that increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Non-lipid polycationicamino polymers may also be used for delivery. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder that may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. Such labeling would include amount, frequency, and method ofadministration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually mice, rabbits, dogs, or pigs. The animal modelmay also be used to determine the appropriate concentration range androute of administration. Such information can then be used to determineuseful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient which ameliorates the symptoms or condition. Therapeuticefficacy and toxicity may be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., ED50 (thedose therapeutically effective in 50% of the population) and LD50 (thedose lethal to 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index, and it can be expressed asthe ratio, LD50/ED50. Pharmaceutical compositions which exhibit largetherapeutic indices are preferred. The data obtained from cell cultureassays and animal studies is used in formulating a range of dosage forhuman use. The dosage contained in such compositions is preferablywithin a range of circulating concentrations that include the ED50 withlittle or no toxicity. The dosage varies within this range dependingupon the dosage form employed, sensitivity of the patient, and the routeof administration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors that may be taken intoaccount include the severity of the disease state, general health of thesubject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc. Pharmaceutical formulations suitable fororal administration of proteins are described, e.g., in U.S. Pat. Nos.5,008,114; 5,505,962; 5,641,515; 5,681,811; 5,700,486; 5,766,633;5,792,451; 5,853,748; 5,972,387; 5,976,569; and 6,051,561.

The following Examples illustrate the present invention, without in anyway limiting the scope thereof.

EXAMPLES

The following methods are employed to perform the examples providedbelow:

Fly Culture

All flies (Drosophila melanogaster, Bloomington Stock center and SzegedStock center) are kept on standard corn meal food according to methodsfamiliar to one of skill in the art. Using conventional methods, allcrosses are done at 25° C. Balancers, or special chromosomes thatprevent recombination and carry at least one dominant morphologicalmarker are used and include CyO, TM3 and TM6B. All flies used carry thew¹¹¹⁸ mutant allele at the white locus. Mutations not referenced hereinand the nomenclatures of standard Drosophila genetics have beendescribed previously and are familiar to one of skill in the art(Lindsley, D. L., and Zimm, G. (1992). The Genome of Drosophilamelanogaster (New York: Academic Press);http://flybase.bio.indiana.edu).

Fly Stocks

The ey-Gal4 (also referred to as “eyeless-Gal4”) strain is obtained fromBloomington stock center (Bloomington, Ill.). In this strain, the yeasttranscription factor Gal4 is expressed in the precursor cells and adultcells of fly eyes. UAS-Dp110^(D954A) and UAS-PKB strains are used(Leevers et al, EMBO J. 1996 Dec. 2;15(23):6584-94; Staveley, et al.Curr Biol. 1998 May 7;8(10):599-602). EP insertion strains are obtainedfrom Bloomington and Szeged stock centers.

Fly Genetic Crosses

The generation of a recombinant second chromosome that bears both theey-Gal4 and UAS-Dp110^(D954A) transgenes is described below: the w;ey-Gal4/CyO flies are crossed to y w; UAS-Dp110^(D954A) flies. Virgindaughters of the genotype w/y w; ey-Gal4/UAS-Dp110^(954A) are crossed tow/Y; Sp/CyO; MKRS,ry/TM2, ry males. A male progeny with the genotypew/Y; ey-Gal4, UAS-Dp110^(D954A)/CyO; MKRS,ry/TM2, ry/+ is selected basedon small eye phenotype and crossed to w/w; Sp/CyO; MKRS, ry/TM2, ryvirgin females to establish w; ey-Gal4, UAS-Dp110^(D954A)/CyO; MKRS,ry/TM2, ry/+ stock.

Crosses Using the X Chromosome EP Insertion Lines

Crosses used for the screening of the X chromosome EP insertion linesare described below: Males of the X chromosome EP insertion lines(EP(X)) are crossed to w, ey-Gal4, UAS-Dp110^(D954A)/CyO; MKRS,ry/TM2,ry/+ females. Adult progenies of the genotype EP(X)/w; ey-Gal4UAS-Dp110^(D954A)/+ and w/Y; ey-Gal4, UAS-Dp110^(D954A)/+ are selectedfor comparison.

Flies are examined under a Zeiss dissecting microscope (Carl ZeissMicroImaging, Inc., Thornwood, N.Y.). The eyes of at least five EP(X)/w;ey-Gal4 UAS-Dp110^(D954A)/+ flies were compared to the eyes of at leastfive w/Y; ey-Gal4, UAS-Dp110^(D954A)/+ (see above). If the average eyesizes of the two fly groups are significantly different upon visualinspection, the EP(X) line is selected as a potential positive.Statistical analysis is then performed by measuring the eye size oflarger number of flies (see below).

Crosses Using the Second Chromosome EP Insertion Lines

The crosses used for the screening of the second chromosome EP insertionlines is described below: Either males or virgin females of the secondchromosome EP insertion lines (EP(2)) are crossed to w, ey-Gal4,UAS-Dp110^(D954A)/CyO MKRS,ry/TM2, ry/+ virgin females or males. Theeyes of at least five ey-Gal4, UAS-Dp110^(D954A)/CyO progeny flies arecompared to the eyes of at least 5 ey-Gal4, UAS-Dp110^(D954A)/EP(2)progeny flies with the same sex. The same criterion was used to selectpotential positive EP(2) lines as in the case of EP(X) lines.Statistical analysis is also used to confirm the positives.

Crosses Using the Third Chromosome EP Insertion Lines

The crosses used for the screening of the third chromosome EP insertionlines is described below: The EP(3) lines obtained from stock centersare balanced either by TM3 or TM6B balancers. Either males or virginfemales of the third chromosome EP insertion lines (EP(3)) are crossedto w; ey-Gal4, UAS-Dp110^(D954A)/CyO; MKRS,ry/TM2, ry/+ virgin femalesor males. The eyes of at least five ey-Gal4, UAS-Dp110^(D954A)/+;EP(3)/+progeny flies are compared to the eyes of at least five ey-Gal4,UAS-Dp110^(D954A)/+; balancer/+progeny flies with the same sex. The samecriterion is used to select potential positive EP(3) lines as in thecase of EP(X) lines. Statistical analysis is used to verify the positivelines.

Statistical Analysis of the Sizes of Fly Eyes

For each respective genotype, twenty fly eyes are photographed using aZeiss dissecting microscope equipped with a Canon digital camera (KodakProfessional DCS 520, Eastman Kodak, Rochester, N.Y.). Images areprocessed by Photoshop 5.0 software (Adobe Systems Incorporated, SanJose, Calif.). To estimate the eye size (two dimensional surface areabased on photograph), an eye is approximated by an oval, thus both thelong (a) and short (b) axis of an eye are measured. The eye size iscalculated as ((a+b)/4)²π. The Student's t-Test (one-tailed, two-sampleunequal variance) is used to test if there is statistically significant(p<0.05) difference between the mean eye size of two genotypes. Beforethe t-Test is applied, the eye size data or its log transformation istested for normality using the Kolmogorov-Smirnov test (Sokal, R. R. andRohif, F. J. (1981) Biometry. W.H.Freeman and Company, New York. 716pp.). SigmaStat for Windows Version 2.03 (SPSS, Inc. Chicago, Ill.) isused for statistical analysis of fly eye size. Because EP(X) insertionis on the X chromosome, the cross EP(X)/Y; +/+ x X/X; ey-Gal4,UAS-Dp110^(D954A)/Balancer produces the progeny with the genotypeEP(X)/X; ey-Gal4, UAS-Dp110^(D954A)/+ but not the genotype X/X;UAS-Dp110^(D954A)/+ needed for internal comparison. Thus, external X/X;UAS-Dp110^(D954A)/+flies are used (see Table 2).

Genetic Assay Development

Two established genetic techniques are used to create the transgenicDrosophila disclosed herein. One is the binary Gal4/UAS system fortissue-specific transcription of a gene In this system, a fly strainexpressing yeast transcription factor GAL4 in tissue X (often referredto as a Gal4 line or a Gal4 driver) is crossed to a fly strain carryinga transgene with GAL4 binding site UAS (upstream activation sequences)fused to gene Y (UAS-Y). Thus, in the progeny, gene Y is expressedspecifically in tissue X.

The other genetic technique used is EP element mutagenesis (Rorth, P.,et al. 1998 Development 125, 1049-57). The EP element is a synthetic,modified P element containing UAS sequences and a basal promoter at oneend. The EP element can randomly insert into the Drosophila genome,often at the 5′ end of a gene. Because the EP element is asymmetric withonly one end containing the UAS sequence and a basal promoter, theeffect of an EP insertion-on nearby genes is influenced by theorientation of the inserted EP element. It has been shown that at theinsertion site, the gene located proximal to the UAS and basalpromoter-containing end of an EP element often has GAL4-dependentexpression However, an EP insertion may also disrupt a cis element ofthe nearby gene and affect its expression in a Gal4-independent manner.A large collection of EP insertion strains are publicly available (seewww.fruitfly.org).

Real Time PCR Analysis of RNA Levels

A N6 random primer (Roche Applied Science, Indianapolis, Ind.) is usedfor the reverse transcription reaction. The primer pairs and probe foreach transcription unit is commercially available from AppliedBiosystems (Foster City, Calif.). Real-time PCR is done using the ABIPrism 7700 Sequence Detection System according to the manufacturer'sinstruction (Applied Biosystems). Primer concentrations are 0.2 pmol/uland the probe concentration is 0.1 uM, in a 25 ul reaction. Cycles are50° C. for 2 min, 95° C. for 10 min, followed by 40 cycles of 95° C. for15 sec, 60° C. for 1 min. Data is analyzed using the relative standardcurve method as described in the User Bulletin#2 Manual for the ABIPrism 7700 Sequence Detection System (Applied Biosystems). All RNAlevels are normalized by using the endogenous RNA levels of theribosomal protein rp49 gene in each sample according to conventionalmethods. For X chromosome EP lines, EP/+ virgin females are crossed tohspGal4/CyO males. Male progenies of the genotype EP/Y; hspGal4/+ arecollected and made to 8 vials with 10 flies per vial. 4 vials are keptat 25° C. and 4 vials are subjected to 6 cycles of heat-shock and restwith each cycle made at 1 hr at 37° C. and 3 hrs at 25° C. Usingconventional methods, total RNAs are prepared from the 10 flies fromeach heat-shocked vial after heat shock and from unheat-shocked vials.Male progenies of the genotype +N; hspGal4/+, the genotype EP/Y; +/CyO,the genotype +N; +/CyO are similarly treated and total RNAs prepared.Thus for each transcription unit assayed of each EP line, the followingdata table is obtained (where hs refers to heat shock and nhs is no heatshock): TABLE 1A RNA levels of a transcription unit Genotype heat-shock(mean value, normalized) EP/Y; hspGal4/+ nhs A EP/Y; hspGal4/+ hs A′+/Y; hspGal4/+ nhs B +/Y; hspGal4/+ hs B′ EP/Y; +/CyO nhs C EP/Y; +/CyOhs C′ +/Y; +/CyO nhs D +/Y; +/CyO hs D′

The Student t-Test is used to determine if there is statisticaldifference between value A and A′. If there is a difference, it wouldindicate that there is a heat shock-dependent effect which may actthrough the interaction between Gal4 and EP. To correct for theindependent effect of heat-shock alone, Gal4 alone and EP alone, theratio of (A′/A)/((B′/B+C′/C+D′/D)/3) is determined and used to indicatethe Gal4-dependent effect of EP on the transcription unit RNA level.(See Table 4, column “Gal4 effect”). The EP insertion may disrupt thefunction of a cis regulatory element of a nearby transcription unit atthe insertion point. This insertion-effect is independent of Gal4. (SeeTable 4, column “Insert effect”). To determine this effect, Studentt-Test is used to determine if there is a statistical difference betweenvalue C and D. If there is a difference, the ration of C/D is used toindicate the insertion-effect of a EP on a transcription unit RNA level.

The same method is used to analyze the second chromosome and thirdchromosome EP lines.

Example 1 Initial Genetic Screen

To generate an easily visible adult phenotype as a basis for geneticmodifier screens to identify proteins involved in the Drosophila insulinsignaling pathway, we sought to disrupt this pathway function in flyeyes. This is because (1) the eye is not essential for flies, (2) theeye phenotype is easily scored, and (3) reducing insulin signaling inthe developing eyes causes a small eye phenotype (Leevers, S J et al,EMBO J. 1996 Dec. 2;15(23):6584-94).

For the small eye phenotype-based genetic modifier screen to work, wereasoned that the phenotype must be modifiable by changing theexpression level of known insulin signaling genes. This is indeed whatwe observed. The small eye phenotype is suppressed by over-expression ofDrosophila PKB via the UAS-PKB transgene, which acts downstream of PI3K.Conversely, the small eye phenotype is also enhanced by over-expressionof PTEN, which acts antagonistically to PI3K (See also Goberdhan, D. C.et al.(1999) Genes Dev 13, 3244-58.; Huang, H., et al., Development 126,5365-72.). These results demonstrate that we should be able to identifynew genes in the insulin signaling pathway by a genetic modifier screenusing the EP insertion strains, which often cause GAL4-dependentover-expression of a nearby gene. These genes can be either positivelyacting (suppressors of the small eye phenotype), or negatively acting(enhancers of the small eye phenotype) in insulin signaling.

We can express a dominant-negative form of Drosophila PI3K catalyticsubunit, Dp110^(D954A), by using the ey-Gal4 transgene which expressesGAL4 in the developing eyes, and the UAS-Dp110^(D954A) transgene(Hazelett, D. J., et al. (1998). Development 125, 3741-51.); Leevers, SJ et al, EMBO J. 1996 Dec. 2;15(23):6584-94).

As disclosed herein, flies carrying a chromosome with both the ey-Gal4and the UAS-Dp110^(D954A) transgenes have smaller eyes as compared towild type control flies. This small eye phenotype has 100% penetrance,indicating strong inhibitory effect of the Dp110^(D954A) protein in theinsulin pathway.

The Drosophila genome contains about 14,000 predicted genes (Adams etal., 2000 Science 287 2185-95). These genes are defined by the predictedtranscripts (sometimes referred to as transcription units or “CeleraTranscripts” (CTs or CGs), see www.Celera.com). Based on the knownfunctions and predicted functions, these genes or transcription unitsare classified into different functional categories or “bins”. Ananalysis of publicly available EP strains revealed 1536 EP strains thathave EP elements inserted within 10 Kb distance upstream from the startcodon (ATG) of 483 genes (CTs). These genes belong to the functionalbins of peptidase, protein kinases/phophatases, signal transductionmolecules, transporters, ligand-binding proteins, transcription factors,enzymes and other types of molecules. The functional bin categories arebased on the Celera Drosophila Jamboree Release 1.

As an initial screen, 112 EP strains that have an EP element insertednext to 68 predicted genes that encode protein kinases/phosphatases ortheir interacting factors are crossed to flies carrying the ey-Gal4UAS-Dp110^(D954A) chromosome and eyes of adult progenies examined asdescribed above. Three EP strains are found to be able to suppress thesmall eye phenotype induced by Dp110^(D954A)-EP(3)3553, EP(X)0382 andEP(3)3459 and are described below:

EP(3)3553/PDK

Statistical analysis confirms that EP(3)3553 is a suppressor ofDp110^(D954A) The mean eye size of the XX; ey-Gal4 UAS-Dp110^(D954A)/+flies is 8.39×10⁻² mm² while the mean eye size of their sisters XX;ey-Gal4 UAS-Dp110^(D954A)/+; EP(3)3553/+ flies is 1.19×10⁻¹ mm². The twomeans are significantly different (p<0.001, Student's t-Test hereafter).The mean eye size of the XY; ey-Gal4 UAS-Dp110^(D954A)/+ flies is alsosmaller that that of their brothers XY; ey-Gal4 UAS-Dp110^(D954A)/+;EP(3)3553/+flies (7.39×10⁻² mm² vs. 9.20×10⁻² mm², p<0.001). Theseresults show that EP(3)3553 suppresses the small eye phenotype caused byDp110^(D954A). See Table 1 below. TABLE 1 EP(3)3553 suppressesDp110^(D954A) 2 1 Mean^(b) 3 4 Genotypes^(a) (eye size, mm²) S.D.^(c)n^(d) 1 XX; ey-Gal4, 1.19 × 10⁻¹ 1.43 × 10⁻² 20 UAS-Dp110^(D954A)/+;EP(3)3553/+ 2 XX; ey-Gal4, 8.39 × 10⁻² 1.60 × 10⁻² 20UAS-Dp110^(D954A)/+; +/+ 3 XY; ey-Gal4, 9.20 × 10⁻² 1.02 × 10⁻² 20UAS-Dp110^(D954A)/+; EP(3)3553/+ 4 XY; ey-Gal4, 7.39 × 10⁻² 1.18 × 10⁻²20 UAS-Dp110^(D954A)/+; +/+^(a)The abbreviated progeny genotypes of the cross X/Y; +/+;EP(3)3553/TM6B, Tb × X/X; ey-Gal4, UAS-Dp110^(D954A)/CyO; +/+. TM6B andCyO are balancer chromosomes (se methods section, above).^(b)Student's t-Test shows that the means of Row 1 and Row 2 differ atthe significant level with p < 0.001. The means of Row 3 and Row 4differ at the significant level with p < 0.001.^(c)S.D. represents standard deviation.^(d)The total number of eyes measured.

EP(3)3553 has an EP element inserted on the third chromosome at the 5′end noncoding sequences of the Drosophila gene Pk61C, which is the flyortholog of mammalian PDK1, Genbank accession number NM_(—)002613(http://flybase.bio.indiana.edu.). PK61C is the only gene found within10 Kb region on both sides of the EP(3)3553 insertion site. The relativeposition of the Pk61C gene to the EP(3)3553 element is such that the 5′end of Pk61C is proximal to the UAS-containing end of the EP(3)3553element (http://flybase.bio.indiana.edu). Thus, it is expected that GAL4would induce the expression of Drosophila PDK1. Since PDK1 is known toact downstream of PI3K in the insulin signaling pathway, it is alsopredicted that over-expression of PDK1 might suppressDp110^(D594A)-induced phenotypes. Thus, given the involvement of PDK1 inthe insulin signaling pathway, the identification of EP(3)3553 as asuppressor of Dp110^(D594A)-induced small eye phenotype stronglysuggests that our genetic screen can be used to identify moleculesrelevant to the insulin pathway.

EP(X)0382/SHP-2

Statistical analysis also confirms that EP(X)0382 significantlysuppresses the small eye phenotype induced by over-expression ofDp110^(D954A). The mean eye size of EP(X)0382/X; ey-Gal4,UAS-Dp110^(D954A)/+ flies (1.04×10⁻¹ mm²) is significantly larger thanthat of the XX; ey-Gal4, UAS-Dp110^(D954A)/+ flies (9.05×10⁻² mm²;p<0.0001), indicating that EP(X)0382 suppresses the function ofDp110^(D954A). See Table 2 below: TABLE 2 EP(X)0382 suppressesDp110^(D954A) 2 3 Mean Mean^(b) (eye (corrected 1 size, eye size, 4 5Genotypes^(a) mm²) mm²) S.D.^(c) n 1 XX; ey-Gal4, 9.05 × 10⁻² 9.05 ×10⁻² 1.06 × 10⁻² 20 UAS-Dp110^(D954A)/+ 2 XY; ey-Gal4, 7.24 × 10⁻² 7.24× 10⁻² 1.04 × 10⁻² 20 UAS-Dp110^(D954A)/+ 3 EP(X)0382/X; 1.06 × 10⁻¹1.04 × 10⁻¹ 9.72 × 10⁻³ 20 ey-Gal4, UAS-Dp110^(D954A)/+ 4 XY; ey-Gal4,7.38 × 10⁻² 7.24 × 10⁻² 9.87 × 10⁻³ 20 UAS-Dp110^(D954A)/+^(a)Row 1 and 2 contain the abbreviated genotypes of the stock w/Y;ey-Gal4, UAS-Dp110^(D954A)/CyO. Rows 3-4 contain the abbreviated progenygenotypes of the cross EP(X)0382/Y; +/+ × w/w; ey-Gal4,# UAS-Dp110^(D954A)/CyO. CyO is a balancer chromosome (see methodssection, above).^(b)Flies in Rows 1-2 are siblings. Flies in Rows 3-4 are siblings. Butflies in Rows 1-2 are not siblings of flies in Rows 3-4. Therefore, theycould have experienced somewhat different growth conditions# which affect the body size including eye size. Thus for the eye sizecomparisons between them to be meaningful, the measured eye sizes ofRows 3-4 are corrected, based on the assumption that the # flies of thecommon genotype XY; ey-Gal4, UAS-Dp11^(D954A)/+ (Row 2, Column 1; Row 4,Column 1) should have the same mean eye size. Each of the 20 eyesmeasured in Rows 3-4 are corrected by the factor 0.981 # (7.24 ×10⁻²/7.38 × 10⁻², see Column 2). This column shows the average of thecorrected eye sizes. Student's t-Test shows that the means of Row 1 andRow 3 differ at the significant level with p < 0.001.^(c)The standard deviation is based on the corrected eye sizes.

EP(X)0382 strain has an EP element inserted on the X chromosome at the5′ end of the Drosophila gene corkscrew (csw), which is the fly orthologof the human protein-tyrosine phosphatase SHP-2 gene (GenBank accessionnumber NM_(—)002834). The insertion site is 201 nucleotides upstream ofthe start site of a csw cDNA (GenBank Accession U19909;www.fruitfly.org). The relative position of the csw gene to theEP(X)0382 element is such that the 5′ end of csw is proximal to thenon-UAS-containing end of the EP(X)0382 element. There is no other genefound within the 10 Kb region on the other side of the EP(X)0382insertion site. Using quantitative real-time RT-PCR,no effect of the EPon csw RNA level was detectable at the level of analysis.

SHP-2 and csw have been shown to play a positive role in receptortyrosine kinase signaling pathways, such as those involving the PDGFreceptor and the EDGP receptor (Allard et al., (1996. Development 122,1137-46; Bennett, A. M., et al. (1994). Proc Natl Acad Sci USA 91,7335-9; Feng, G. S. et al. (1993) Science 259, 1607-11; Perkins, L.A.,et al., (1996). Dev Biol 180, 63-81; Vogel, W.,et al. 1993 Science259, 1611-4; .Xiao, S., et al. (1994) J Biol Chem 269, 21244-8).

SHP-2 has also been implicated in insulin signaling, although whether itplays a positive or negative role is presently unknown (Kharitonenkov,A. et al., (1995). J Biol Chem 270, 29189-93; Maegawa, H., et al.(1999)J Biol Chem 274, 30236-43.; Myers, M. G., et al. (1998) J Biol Chem 273,26908-14; Ugi, S., et al. J Biol Chem 271, 12595-602).

Since SHP-2 is a known gene in the mammalian insulin signaling pathway,it suggests that the modifier screen disclosed herein may be used toidentify molecules relevant to human diabetes.

EP(3)3459/KIAA0336

The suppression of the Dp110^(D954A)-induced small eye phenotype byEP(3)3459 is statistically significant. In both XX and XY flies, thepresence of EP(3)3459 insertion increases the mean eye size of ey-Gal4,UAS-Dp110^(D954A)/+ flies (9.71×10⁻² mm² versus 9.09×10⁻² mm² in XXflies, p<0.05; 8.10×10⁻² mm² versus 7.13×10⁻² mm² in XY flies, p<0.003).See Table 3 below. TABLE 3 EP(3)3459 suppresses Dp110^(D954A) 2 1Mean^(b) 3 4 Genotypes^(a) (eye size, mm²) S.D. n 1 XX; ey-Gal4, 9.71 ×10⁻² 1.14 × 10⁻² 20 UAS-Dp110^(D954A)/+; EP(3)3459/+ 2 XX; ey-Gal4, 9.09× 10⁻² 1.10 × 10⁻² 20 UAS-Dp110^(D954A)/+; +/+ 3 XY; ey-Gal4, 8.10 ×10⁻² 8.75 × 10⁻³ 20 UAS-Dp110^(D954A)/+; EP(3)3459/+ 4 XY; ey-Gal4, 7.13× 10⁻² 1.03 × 10⁻² 20 UAS-Dp110^(D954A)/+; +/+^(a)The abbreviated progeny genotypes of the cross X/Y; +/+;EP(3)3459/TM6B, Sb, Tb × X/X; ey-Gal4, UAS-Dp110^(D954A)/CyO; +/+. TM6Band CyO are balancer chromosomes (see methods section above)^(b)Student's t-Test shows that the means of Row 1 and Row 2 differ atthe significant level with p < 0.05. The means of Row 3 and Row 4 differat the significant level with p < 0.003.

EP(3)3459 strain has an EP element inserted on the third chromosome atabout 5 Kb upstream from the 5′ end of the Drosophila casein kinaseII-alpha subunit interactor-3 (CkII-α-i3, Genbank accession numberAF090440). The 5′ end of CkII-α-i3 is proximal to the non-UAS-containingend of the EP(3)3459 element. On the other side of the EP(3)3459insertion site, no gene was found within the 10 Kb region. QuantitativeRT-PCR (Taqman) experiments indicate that this EP element causes 4 foldloss-of-expression of CkII-α-i3.

The human homolog of CkII-α-i3 is suspected to be the human KIAA0336gene (GenBank accession number AB002334) Nagase, T., et al. (1997) DNARes 4, 141-50.

The KIAA0336 gene encodes a protein with 1583 amino acid residues withno known function. The cDNA that defines KIAA0336 was derived from braintissue. The KIAA0336 gene is expressed in all tissue categories exceptthe stomatognathic system based on LifeSeq®Gold 5.1. It has the highestexpression in the hemic and immune system, as judged by the percentageof KIAA0336 gene-derived cDNA clones among the total number of sequencedcDNA clones from the hemic and immune system. The apparent lack of theKIAA0336 gene-derived cDNA clones in the stomatognathic system librariesmay be due to the lower number of sequenced cDNA clones (10,988) fromthe libraries of the stomatognathic system as compared to 648,215sequenced clones from the libraries of the hemic and immune system.

CkII-α-i3 was identified through a two-hybrid interaction with CKII αsubunit (see also the Genbank annotation, Genbank accession numberAF090440). It encodes a putative nuclear protein with CKII consensusphosphorylation sites. While the function of CkII-α-i3 has not beenreported, CKII is likely involved in the insulin signaling pathway basedon data which shows that a CKII inhibitor blocks the insulin-inducedDNA-binding activity of nuclear extracts and the CKII α subunitinteracts with the insulin receptor substrate-1 (IRS-1) in vitro (Kim,S. J., and Kahn, C. R. (1997) Biochem J 323, 621-7.; Li et al., 1999).It is contemplated herein that the human ortholog of CkII-α-i3,KIAA0336, plays a role in the insulin signaling pathway, and as such isa suitable drug target for the development of therapeutics to treat,prevent or ameilorate pathological conditions associated with thedysregulation of the insulin signaling pathway.

Thus, we have expressed a dominant negative form of the Drosophilaphosphoinositide 3-kinase (PI3K) in developing eye. This results in asmall eye phenotype in adult flies. The small eye phenotype issuppressed by over-expression of protein kinase B (PKB), which actsdownstream of PI3K in the insulin signaling pathway (Lasko, P. ClinGenet. 2002 November;62(5):358-67), suggesting that the small eyephenotype can be used as an assay for a genetic modifier screen. Ourstudies identified three mutations that suppress the small eyephenotype. One mutation is linked to the Drosophila ortholog of the3′-phosphoinositide-dependent kinase 1 gene (PDK1). Another mutation islinked to the Drosophila ortholog of the protein-tyrosine phosphataseSHP-2 gene. Both PDK1 and SHP-2 are known genes in the mammalian insulinsignaling pathway, suggesting that the screen will identify moleculesrelevant to human diabetes. The third mutation is linked to theDrosophila gene CkII-α-i3, whose human ortholog is KIM0336.

Example 2 Identification of Proteins

Using methods described above, proteins were identified which aresuspected to play a role in the insulin signaling pathway. These genesand their human homologs (listed in column “best hu”) (as well asKIAA0336, discussed above) are set forth below in Table 4. The geneticscreen identified the suppressors, which were divided into categoriesdepending on strength of suppression, where S1 is the strongest and S4the weakest, ND is not determined. TABLE 4 Modifiers Best hu CeleraGal4- insert- blastp E Gene EP females males transcript effect effectbest hu value FMR2 protein EP(2)2172 S1 S1 CG8817(lilli) no 0.53×  NM_002025 1.E−14 nucleoside diphosphatase (ER-UDPase) EP(2)2172 S1 S1CT10292 2.2×  0.79×   inositol hexkisphosphate kinase 3 EP(2)2440 S1 S1CT28369 2.7×  0.60×   NM_054111 5.E−39 RNA polymerase I 16Kd subunitEP(2)2483 S2 S1 CT29944/CG10685 70× 1.34×   brain tumor EP(2)2483 S2 S1CG10719 NM_033279.1 5.E−37 neuroligin 3 EP(2)2615 S1 S1 CT12983 5.5×  noNM_018977 7.E−68 retinoblastoma-binding protein 5 EP(3)3121 S1 S1CT17654 35× 1.9×  NM_005057.1 0.E+00 polypeptide chain release factor 1EP(3)3121 S1 S1 CT17696 no 0.78×   CYSTEINE STRING PROTEIN (CSP)EP(3)3141 S1 S2 CT19114 no 0.5×  S70516 7.E−40 hypothetical proteinEP(3)3141 S1 S2 CT36401 13× no NM_0.16472 6.E−10 TRANSCRIPTION FACTORADF-1 (ADH EP(3)3354 S1 S1 CT16251 36× no none DISTAL FACTOR 1) membraneprotein TMS-2 EP(3)3355 S1 S1 CT15063  7× no AB033079.1 6.E−90 unknownEP(3)3355 S1 S1 CT14836(failed no 0.05×   XM_046613.1 4.E−22 axonconnection) 3-phosphoinositide dependent protein EP(3)3553 S1 S1 CT42509140×  kinase-1 hypothetical protein EP(3)3628 S1 S1 CT5336 10× noNM_015343.1 6.E−37 SH3 domain-containing protein SH3d19 EP(3)3634 S1 S1CT22037 2.4×  no XM_037453 1.E−12 poly (rC)-binding protein 3 EP(3)3638S1 S2 contig_4403_1 NM_020528.1 1.E−76 nucleolar RNA-associated proteinalpha EP(3)3652 S1 S2 CT37030 NM_022917 1.E−98 Casein kinase I, gamma 3EP(3)3652 S1 S2 gish NM_004384.1  1.E−170 acyl-Coenzyme A dehydrogenase,EP(3)3660 S2 S1 CT12987 no 0.60×   NM_001609  1.E−142 short/branchedchain precursor (ACADSB) ribosomal protein L26 EP(3)3660 S2 S1 CT212071.6×  no myosin phosphatase target subunit 1 EP(3)3723 S1 S1 CT184154.5×  no NM_002480 8.E−73 GPI transamidase EP(X)0427 S1 S1 CT14360 24×1.6×  XM_039644  1.E−118 no good hu/mo homologs EP(X)0427 S1 S1 CT14236no no mouse preprocathepsin B EP(X)1101 S2 S1 CT30795 no 0.54×  NM_001908.1  1.E−106 no good hu/mo homologs EP(X)1101 S2 S1 CT310777.8×  no tousled-like kinase 2/SNARE protein EP(X)1413 S1 S1CG2829/CG12462 NM_006852.1  1.E−146 kinase SNAK CKII-alpha-I3 (KIAA0336)EP(3)3459 S5 S5 CG3217 no 0.26×   AB002334 2.3E−6  glycogenin glucosyltransferase activity EP(2)2045 S2 S2 CG9480 ND ND NM_004130.2 3.00E−93 CO monooxygenase EP(2)2399 S2 S2 CG8776 ND ND NM_024843.2 9.00E−26  COmonooxygenase EP(2)2399 S2 S2 CG8768 ND ND NM_020195.1 2.00E−57  Na-Ktransporter EP(2)2454 S1-2 S2 CG9258 ND ND NM_001677.1 2.00E−33  DNAbinding protein transglycosidases EP(3)3097 S2 S2 CG7187 ND NDNM_018070.2 5.00E−47  DNA binding protein transglycosidases EP(3)3097 S2S2 CG7985 ND ND NM_173620.1 1.00E−43  chaperone activity EP(3)3141 S1 S2CG6395 ND ND NM_173650.1 3.00E−25  ubiquitin specific protease EP(3)3187S2 S3 CG5486 ND ND NM_0179442 1.00E−144  SH3/SH2 adaptor protein SAM,pointed EP(3}3474 S3 S2 CG31163 ND ND NM_015278.1 3.00E−31  domaincasein kinase I, gamma 3 activity EP(3)3652 S1 S2 CG6963 ND NDNM_004384.1 1.00E−170  AcylCoA dehydrogenase EP(3)3660 S2 S1 CG3902 NDND NM_001609.1 1.00E−141  hexokinase Lysophosphatidic add EP(X)0352 S1-2S2 CG3001 ND ND NM_00189.3 1.00E−119  acyltransferase hexokinaseLysophosphatidic acid EP(X)0352 S1-2 S2 CG32699 ND ND NM_017839.12.00E−68  acyltransferase carboxypeptidase H EP(X)0356 S2 S2 CG4122 NDND NM_001304.3 0 sec. carrier membrane protein EP(X)1007 S3 S2 CG9195 NDND NM_004866.2 1.00E−64  adenosylhomocysteinase sec. carrier membraneprotein EP(X)1007 S3 S2 CG11654 ND ND NM_000687.1 0adenosylhomocysteinase a/b hydrolase EP(X)1101 S2 S1 CG33174 ND NDNM_006133.1 0 FMR2 protein EP(2)2172 S1 S1 CG8817(lilli) no 0.53×  NM_002025 1.E−14 nucleoside diphosphatase (ER-UDPase) EP(2)2172 S1 S1CT10292 2.2×  0.79×   inositol hexkisphosphate kinase 3 EP(2)2440 S1 S1CT28369 2.7×  0.60×   NW_054111 5.E−39 RNA polymerase I 16Kd subunitEP(2)2483 S2 S1 CT29944/CG10685 70× 1.34×   brain tumor EP(2)2483 S2 S1CG10719 NM_033279.1 5.E−37 neuroligin 3 EP(2)2615 S1 S1 CT12983 5.5×  noNM_018977 7.E−68 retinoblastoma-binding protein 5 EP(3)3121 S1 S1CT17654 35× 1.9×  NM_005057.1 0.E+00 polypeptide chain release factor 1EP(3)3121 S1 S1 CT17696 no 0.78×   CYSTEINE STRING PROTEIN (CSP)EP(3)3141 S1 S2 CT19114 no 0.5×  S70516 7.E−40 hypothetical proteinEP(3}3141 S1 S2 CT36401 13× no NM_016472 6.E−10 TRANSCRIPTION FACTORADF-1 (ADH EP(3)3354 S1 S1 CT16251 36× no none DISTAL FACTOR 1) membraneprotein TMS-2 EP(3)3355 S1 S1 CT15063  7× no AB033079.1 6.E−90 unknownEP(3)3355 S1 S1 CT14836(failed no 0.05×   XM_046613.1 4.E−22 axonconnection) 3-phosphoinositide dependent protein EP(3)3553 S1 S1 CT42509140×  kinase-1 hypothetical protein EP(3)3628 S1 S1 CT5336 10× noNM_015343.1 6.E−37 SH3 domain-containing protein SH3d19 EP(3)3634 S1 S1CT22037 2.4×  no XM_037453 1.E−12 poly (rC)-binding protein 3 EP(3)3638S1 S2 contig_4403_1 NM_020528.1 1.E−76 nucleolar RNA-associated proteinalpha EP{3)3652 S1 S2 CT37030 NH_022917 1.E−98 Casein kinase I, gamma 3EP(3)3652 S1 S2 gish NM_004384.1  1.E−170 acyl-Coenzyme A dehydrogenase,EP(3)3660 S2 S1 CT12987 no 0.60×   NM_001609  1.E−142 short/branchedchain precursor (ACADSB) ribosomal protein L26 EP(3)3660 S2 S1 CT212071.6×  no myosin phosphatase target subunit 1 EP(3)3723 S1 S1 CT184154.5×  no NM_002480 8.E−73 GPI transamidase EP(X)0427 S1 S1 CT14360 24×1.6×  XM_039644  1.E−118 no good hu/mo homologs EP(X)0427 S1 S1 CT14236no no mouse preprocathepsin B EP(X)1101 S2 S1 CT30795 no 0.54×  NM_001908.1  1.E−106 no good hu/mo homologs EP(X)1101 S2 S1 CT310777.8×  no tousled-like kinase 2/SNARE protein EP(X)1413 S1 S1CG2829/CG12462 NM_006852.1  1.E−146 kinase SNAK CKII-alpha-i3(KIAA0336)EP(3)3459 S5 S5 CG3217 no 0.26×   AB002334 2.3E−6 

To verify that a protein (gene listed in Table 4) (i.e. “a modifiergene”) is involved in insulin signaling and metabolism, one may examineif the perturbation of a modifier gene will cause a change in theexpression level of an insulin responsive sequence (IRS)-controlledreporter gene. It is known that the activation of the insulin signalingpathway leads to the inhibition of the forkhead transcription factorsthat bind to the insulin responsive sequences. Thus, changes in theinsulin signaling level by a modifier may lead to changes in theexpression level of the IRS-controlled genes. In addition, because theinsulin signaling pathway controls glucose and lipid metabolism, one mayexamine if the perturbation of a modifier gene causes a change in theglycogen or lipid level in the fly.

The genetic screen is designed to identify both genes that activate andthe genes that repress the insulin signaling pathway. However, becausestimulating the insulin signaling pathway is needed for the treatment ofdiabetes, the genes that normally inhibit insulin signaling may be thepreferred drug targets.

Example 3 Suppression Modification Screen

In addition to the genetic screen discussed above which is based on thedominant phenotype induced by a dominant-negative form of fly PI3K(noted from now on as Dp110^(DN)), a similar genetic screening methodmay also be developed using an additional known gene in the insulinsignaling pathway, PTEN, a negative regulator of the ISP.

Over-expression of PTEN in developing eyes also causes a small eyephenotype, and the eye phenotype observed following Dp110^(DN)overexpression in the eye is further enhanced by over-expression ofPTEN. This double-mutant transgenic fly stock, capable of overexpressionof both Dp110^(DN) and PTEN is maintained in the presence of a tub:Gal80transgene. The constitutive expression of Gal80 from this transgenebinds to Gal4 protein, blocking Gal4-mediated transcriptionalactivation. This in turn stops the accumulation of genetic modifiersthat ameliorate the reduction in viability associated withoverexpression of components of the ISP.

Based on the additive effect of the two phenotypes, a suppressor screenmay be established. The enhanced small eye phenotype is better suitedfor a screen in which the expected 2-fold reduction in gene productassociated with heterozygosity for loss-of-function mutations wouldproduce a noticeable suppression of the phenotype, i.e. insertionmutations that would be haplo-insufficient with respect to thesensitized ISP phenotype resulting from co-overexpression of bothDp110^(DN) and PTEN.

An analysis of publicly available P-element mutations reveal that thereare over two thousand P-element insertions that reside within a geneticregion halting gene product expression. A fly stock that contains one ofthese P-elements is said to have a 50% decrease of genetic expression.The P-elements screened are of two types. The first type of P-elementcontains a mini white gene(mw) and results in a red eye phenotype whenexpressed in a white 1118 background. The second type of P-elementcontains a mini rosy gene (rw) that when expressed in a white plus androsy minus background results in a rosy eye phenotype. The fly stocksthat contain the eyGal4, UAS Dp110^(DN), UASPTEN, and tubGal80 must becontained within the two backgrounds so that the P-elements can bewitnessed when introduced.

Creation of the Recombinant Stock

The eyGal4, UAS Dp110^(DN), and UASPTEN transgenes all reside on thesecond chromosome and may be recombined together as shown in FIG. 1. Thecreation of the w; ey-Gal4, UAS-Dp110^(D954A)/CyO; MKRS,ry/TM2, ry/+stock previously described. This stock was then recombined with UAS-PTENin a white 1118 background as well as the rosy minus background usingstandard drosophila genetics.

The Introduction of tub-Gal80

Tub-Gal80 can be accessed publicly through the Bloomington Stock Centre.The transgene mutant available is on the X chromosome. The transgene istransposed to a new position on the CyO Balancer Chromosome usingstandard drosophila transposition crosses. The tub-Gal80 CyO is then putinto the white 1118 background as well as the rosy minus background andbalanced over Sco second balancer chromosome. The tub-Gal80 CyO stocksare then crossed with the eyGal4, UAS Dp110^(DN), UASPTEN/CyO stockscreating eyGal4, UAS Dp110^(DN), UASPTEN/tub-Gal80 CyO. These are donein the white 1118 background as well as the rosy minus background.

For screens of the X chromosome homozygous lethal P-element insertions,fly crosses are shown in FIG. 2.

For screens of the second chromosome homozygous lethal P-elementinsertions, fly crosses are shown in FIG. 3.

For screens of the third chromosome homozygous lethal P-elementinsertions, fly crosses are shown in FIG. 4.

Example 4 Identification of Additional Proteins

Using methods described above, additional proteins were identified fromwhich are suspected to play a role in the insulin signalling pathway.The following P-element insertions gave a suppression effect to thesmall eye phenotype caused by over-expression of Dp110^(DN) and PTEN.These genes and their human homologs are set forth below in Table 5.TABLE 5 Modifiers P Human Fly Gene Function element CG Homolog HumanName

CG9187 unknown 10156 CG9187 NM_021067 KIAA0186 gene product

Sac1 phosphoinositide phosphatase 10156 CG9128 NM_014016 suppressor ofactin 1

Dsac1 Gprk2 G protein-coupled receptor 10351 CG17998 NM_005308 Gprotein-coupled

kinase 2 receptor kinase 5 mei-P19 unknown 11592 CG9924 NM_003563speckle-type POZ protein

CG7134 protein tyrosine/serine/ 12038 CG7134 NM_033313 CDC14 homolog A

threonine phosphatase fringe acetylglucosaminyltransferase; 12109CG10580 NM_002405 manic fringe homolog

transferase, transferring glycosyl groups; UDP- glycosyltransferaseCG31132 unknown 12155 CG31132 NM_017934 pleckstrin homology domain

interacting protein ariadne 2 ubiquitin-protein ligase 12341 CG5709NM_006321 ariadne homolog 2; all-trans

retinoic acid inducible RING finger CG5429 unknown 11487 CG5429NM_003766 beclin 1

CG5991 phosphatidylserine 11487 CG5991 NM_014338 phosphatidylserine

decarboxylase decarboxylase CG5261 dihydrolipoamide S- 12171 CG5261NM_001931 dihydrolipoamide

acetyltransferase S-acetyltransferase

Example 6 Additional Modifier Screens

In addition to the genetic screens discussed above which is based on thedominant phenotype induced by a dominant-negative form of fly PI3K, newgenetic screening methods may also be developed using other known genesin the insulin signaling pathway. Screens such as those described aboveare especially desirable since they are a “selection”-based screen.Furthermore, a fluorescent reporter-based genetic screen may also beused. The advantage of this approach is that it is adaptable for highthroughput automation. For example, it is known that the insulin pathwaycontrols the activity of the FKHR-type forkhead transcription factors. Afluorescent reporter can be made using the forkhead binding sites tocontrol flourescent marker expression. Flies carrying this reporterscreened with a fluorescent Drosophila larvae sorter would be anefficient system to identify new genes in the insulin pathway

1. A method to treat, prevent or ameliorate pathological conditionsassociated with dysregulation of the insulin signaling pathwaycomprising administering to a subject in need thereof an effectiveamount of a modulator of a protein selected from the group consisting ofthose disclosed in Table 4 or Table
 5. 2. The method of claim 1 whereinsaid condition is Type 11 diabetes.
 3. The method of claim 1 whereinsaid condition is the Type A syndrome of insulin resistance.
 4. Themethod of claim 1 wherein said modulator inhibits the biochemicalfunction of said protein in said subject.
 5. The method of claim 4wherein said modulator comprises one or more antibodies to said protein,or fragments thereof, wherein said antibodies or fragments thereof caninhibit the biochemical function of said protein in said subject.
 6. Themethod of claim 1 wherein said modulator enhances the biochemicalfunction of said protein in said subject.
 7. The method of claim 1wherein said modulator inhibits gene expression of said protein in saidsubject.
 8. The method of claim 7 wherein said modulator comprises anyone or more substances selected from the group consisting of antisenseoligonucleotides, triple helix DNA, ribozymes, RNA aptamers, siRNA,double stranded RNA and single stranded RNA wherein said substances aredesigned to inhibit gene expression of said protein.
 9. The method ofclaim 1 wherein said modulator enhances the gene expression of saidprotein in said subject.
 10. A method to treat, prevent or amelioratepathological conditions associated with dysregulation of the insulinsignaling pathway comprising administering to a subject in need thereofa pharmaceutical composition comprising an effective amount of amodulator of a protein selected from the group consisting of thosedisclosed in Table 4 or Table
 5. 11. The method of claim 10 wherein saidcondition is Type II diabetes.
 12. The method of claim 10 wherein saidcondition is the Type A syndrome of insulin resistance.
 13. The methodof claim 10 wherein said modulator inhibits the biochemical function ofsaid protein in said subject.
 14. The method of claim 13 wherein saidmodulator comprises one or more antibodies to said protein, or fragmentsthereof, wherein said antibodies or fragments thereof can inhibit thebiochemical function of said protein.
 15. The method of claim 10 whereinsaid modulator enhances the biochemical function of said protein in saidsubject
 16. The method of claim 10 wherein said modulator inhibits geneexpression of said protein in said subject.
 17. The method of claim 16wherein said modulator comprises any one or more substances selectedfrom the group consisting of antisense oligonucleotides, triple helixDNA, ribozymes, RNA aptamers, siRNAr double stranded RNA and singlestranded RNA wherein said substances are designed to inhibit geneexpression of said protein.
 18. The method of claim 10 wherein saidmodulator enhances gene expression of said protein in said subject. 19.A method to identify modulators useful to treat, prevent or amelioratepathological conditions associated with dysregulation of the insulinsignaling pathway comprising assaying for the ability of a candidatemodulator to modulate the biochemical function of a protein selectedfrom the group consisting of those disclosed in Table 4 or Table
 5. 20.The method of claim t 9 wherein said method further comprises assayingfor the ability of an identified modulator to reverse the pathologicaleffects observed in animal models of said conditions.
 21. The method ofclaim 19 wherein said method further comprises assaying for the abilityof an identified modulator to reverse the pathological effects observedin clinical studies with subjects with said conditions.
 22. The methodaccording to claim 19 wherein said condition is Type II diabetes. 23.The method according to claim 19 wherein said condition is the Type Asyndrome of insulin resistance.
 24. A method to identify modulatorsuseful to treat, prevent or ameliorate pathological conditionsassociated with dysregulation of the insulin signaling pathwaycomprising assaying for the ability of a candidate modulator to modulategene expression of a protein selected from the group consisting of thosedisclosed in Table 4 or Table
 5. 25. The method according to claim 24wherein said method further comprises assaying for the ability of anidentified inhibitory modulator to reverse the pathological effectsobserved in animal models of said condition.
 26. The method according toclaim 24 wherein said method further comprises assaying for the abilityof an identified inhibitory modulator to reverse the pathologicaleffects observed in clinical studies with subjects with said condition.27. The method according to claim 24 wherein said condition is Type IIdiabetes.
 28. The method according to claim 24 wherein said condition isthe Type A syndrome of insulin resistance.
 29. A pharmaceuticalcomposition comprising a modulator to a protein selected from the groupconsisting of those disclosed in Table 4 or Table 5 in an amounteffective to treat, prevent or ameliorate pathological conditionsassociated with dysregulation of the insulin signaling pathway in asubject in need thereof.
 30. The pharmaceutical composition according toclaim 29 wherein said condition is Type II diabetes.
 31. Thepharmaceutical composition according to claim 29 wherein said conditionis the Type A syndrome of insulin resistance.
 32. The pharmaceuticalcomposition according to claim 29 wherein said modulator inhibits thebiochemical function of said protein.
 33. The pharmaceutical compositionof claim 29 wherein said modulator comprises one or more antibodies tosaid protein, or fragments thereof, wherein said antibodies or fragmentsthereof can inhibit the biochemical function of said protein.
 34. Thepharmaceutical composition according to claim 29 wherein said modulatorenhances the biochemical function of said protein.
 35. Thepharmaceutical composition according to claim 29 wherein said modulatorinhibits gene expression of said protein.
 36. The pharmaceuticalcomposition of claim 29 wherein said modulator comprises any one or moresubstances selected from the group consisting of antisenseoligonucleotides, triple helix DNA, ribozymes, RNA aptamer, siRNA,double stranded RNA and single stranded RNA wherein said substances aredesigned to inhibit gene expression of said protein.
 37. Thepharmaceutical composition according to claim 25 wherein said modulatorenhances gene expression of said protein.
 38. A method to diagnosesubjects suffering from pathological conditions associated withdysregulation of the insulin signaling pathway who may be suitablecandidates for treatment with modulators to a protein selected from thegroup consisting of those disclosed in Table 4 or Table 5 comprisingassaying mRNA levels of any one or more of said proteins in a biologicalsample from said subject wherein subjects with altered levels comparedto controls would be suitable candidates for modulator treatment.
 39. Amethod to diagnose subjects suffering from pathological conditionsassociated with dysregulation of the insulin signaling pathway who maybe suitable candidates for treatment with modulators to a proteinselected from the group consisting of those disclosed in Table 4 orTable 5 comprising detecting levels of any one or more of said proteinsin a biological sample from said subject wherein subjects with alteredlevels compared to controls would be suitable candidates for modulatortreatment.
 40. A method to treat, prevent or ameliorate a pathologicalcondition associated with dysregulation of the insulin signaling pathwaycomprising: (a) assaying for mRNA levels of a protein selected from thegroup consisting of those disclosed in Table 4 or Table Sin a subject;and, (b) administering to a subject with altered levels of mRNA of saidprotein compared to controls a modulator to said protein in an amountsufficient to treat, prevent or ameliorate the pathological effects ofsaid condition.
 41. The method of claim 40 wherein said condition isType II diabetes.
 42. The method of claim 40 wherein said condition isthe Type A syndrome of insulin resistance.
 43. The method of claim 40wherein said modulator enhances the gene expression of said protein. 44.The method of claim 40 wherein said modulator inhibits the geneexpression of said protein.
 45. A method to treat, prevent or amelioratea pathological condition associated with dysregulation of the insulinsignaling pathway comprising: (a) assaying for levels of a proteinselected from the group consisting of those disclosed in Table 4 orTable Sin a subject; and, (b) administering to a subject with alteredlevels of said protein compared to controls a modulator to said proteinin an amount sufficient to treat, prevent or ameliorate the pathologicaleffects of said condition.
 46. The method of claim 45 wherein saidcondition is Type II diabetes.
 47. The method of claim 45 wherein saidcondition is the Type A syndrome of insulin resistance.
 48. The methodof claim 45 wherein said modulator enhances the biochemical function ofsaid protein.
 49. The method of claim 45 wherein said modulator inhibitsthe biochemical function of said protein.
 50. A diagnostic kit fordetecting mRNA levels of a protein selected from the group consisting ofthose disclosed in Table 4 or Table 5 in a biological sample, said kitcomprising: (a) a polynucleotide of a polypeptide set forth in Table 4,Table 5 or fragments thereof; (b) a nucleotide sequence complementary tothat of (a); (c) a polypeptide of Table 4 or Table 5 of the presentinvention encoded by the polynucleotide of (a), (d) an antibody to thepolypeptide of (c) (e) an RNAi sequence complementary to that of (a)wherein components (a), (b), (c), (d) or (e may comprise a substantialcomponent.
 51. A diagnostic kit for detecting levels of a proteinselected from the group consisting of those disclosed in Table 4 orTable 5 in a biological sample, said kit comprising: (a) apolynucleotide of a polypeptide set forth in Table 4, Table 5 orfragments thereof; (b) a nucleotide sequence complementary to that of(a); (c) a polypeptide of Table 4 or Table 5 of the present inventionencoded by the polynucleotide of (a), (d) an antibody to the polypeptideof (c) (e) an RNAi sequence complementary to that of (a) whereincomponents (a), (b), (c), (d) or (e) may comprise a substantialcomponent.
 52. A method to identify genetic modifiers of the insulinsignaling pathway, said method comprising (a) providing a transgenic flywhose genome comprises a DNA sequence encoding a polypeptide comprisingDp110^(D954A)., said DNA sequence operably linked to a tissue specificcontrol sequence, and expressing said DNA sequence, wherein expressionof said DNA sequence results in said fly displaying a transgenicphenotype; (b) crossing said transgenic fly with a fly containing amutation in a known or predicted gene; and (c) screening progeny of saidcrosses for flies that carry said DNA sequence and said mutation anddisplay modified expression of the transgenic phenotype as compared tocontrols.
 53. The method of claim 52 wherein said DNA sequence encodesDp110^(D954A) and wherein said tissue specific expression controlsequence comprises the eye specific enhancer, ey-Gal4.
 54. The method ofclaim 53 wherein expression of said DNA sequence results in said flydisplaying the “small eye” phenotype.
 55. A method to identify targetsfor the development of therapeutics to treat, prevent or amelioratepathological conditions associated with dysregulation of the insulinsignaling pathway said method comprising identifying the human homologsof the genetic modifiers identified according to the method of claim 52.56. The method of claim 55 wherein said condition is Type II diabetes.57. The method of claim 55 wherein said condition is the Type A syndromeof insulin resistance.